THE CHEMILUMINESCENCES AND DEGRADATIONS OF p - LACTAM
ANTIBIOTICS AFTER OXIDATION BY POTASSIUM SUPEROXIDE
By
JINGSHUN SUN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998
...J
Copyright 1998
by
Jingshun Sun
This work is dedicated to my parents, and my wife, Shuang Yang.
ACKNOWLEDGMENTS
I wish to offer my sincere thanks and gratitude to the chairman of my committee,
my mentor Dr. John H. Perrin, whose friendship, support and unending patience has
made my graduate career a very enjoyable experience.
I thank all of the graduate students in Medicinal Chemistry department for their
interest in my work, suggestions and friendship. Thanks go to Dr. James Winefordner,
Dr. Ian Tebbett, and Dr. Ray Bergeron for their enthusiasm and consistent support of this
project. Special thanks go to Dr. Kenneth Sloan and Dr. Stephen Schuhnan for their
helpfiil advice and friendship over the last several years.
I would like to thank my family for their love and support especially my parents;
they deserve much of the credit for making me the person I am today. Thanks to Jan
Kallman and Nancy Rosa who helped to make Florida my home. I would also like to
extend my thanks to all faculty, students and stuff at Chemistry department whose
assistance is an indispensable part of this achievement. Lastly, I would like to offer my
thanks to Shuang Yang for her love, patience and perseverance.
IV
TABLE OF CONTENTS
; ' page
ACKNOWLEDGMENTS iv
UST OF TABLES : viii
LIST OF FIGURES x
ABSTRACT xvii
CHAPTERS
1 INTRODUCTION 1
Reactive Oxygen Species 1
Singlet Oxygen 1
Superoxide Anion 4
Oxygen Toxicity and Diseases 6
Rheumatoid Arthritis 6
Pulmonary Emphysema 7
Ischemic-Reperfusion Tissue Injury 8
Carcinogenesis 8
Aging 9
Neurodegenerative Disorders 10
Chemotherapy 11
Removal of Free Radicals by Superoxide Dismutase and Vitamin E 11
Increasing Formation of Free Radicals 12
Chemiluminescence and P-Lactam Antibiotics 14
p-Lactam Antibiotics 14
Chemiluminescence 15
2 OBJECTIVES 32
3 CHEMILUMINESCENT METHODOLOGY OF STATIC MODE 36
Materials and Apparatus 36
Experimental Methods 37
Preparations of Stock Solutions 38
Preparation of Luminol Solution 38
Preparation of Hydrogen Peroxide Solution 39
Preparation of Saturated Potassium Superoxide Solution 39
Preparation of Phosphate Buffer Solutions 39
4 THE CHEMICAL PROPERTIES OF POTASSIUM SUPROXIDE 47
Comparison Studies 47
Solvent Selection for Potassium Superoxide 48
5 CHEMILUMINESCENCES AND p-LACTAM ANTIBIOTICS 54
Introduction 54
Probe of Maximum Emission Wavelength 55
Discussion 55
6 MECHANISTIC STUDIES 65
The Deuteration Experiment 65
NMR Consideration 69
NMR Spectra of Penicillin G 70
NMR Spectra of Penicillin V 72
Conclusion 74
TLC Analysis 74
Introduction 74
Experimental 75
Results and Discussions 76
HPLC Detection 78
Experimental 78
Results and Discussion 80
Conclusion 80
HPLC/ESI-MS and HPLC/APCI-MS Analysis of the Degradation of
Penicillin G Following the Oxidation by Potassium Superoxide 81
Introduction 81
Experimental 84
Results and Discussion 85
7 SUMMARY 120
GLOSSARY ..:..... :.......:. 123
VI
APPENDICES
A 124
B '. 142
C 173
D ...! 186
E 194
REFERENCE 199
BIOGRAPHICAL SKETCH 207
vii
LIST OF TABLES
Table page
Table 1-1: Some deleterious effects of systems generating the superoxide radical 28
Table 1-2: Neurodegenerative disorders associated with free radicals 29
Table 1-3: Family of p-lactam antibiotics 30
Table 1-4: Analytical useful chemiluminescent emitters 31
Table 3-1: Chemicals and reagents utilized in the static studies of chemiluminescence...41
Table 3-2: Chemical structures and sources of p-lactam antibiotics examined 42
Table 3-2~continued 43
Table 3-2 — continued 44
Table 3-3: The composition of phosphate buffer solutions 45
Table 6-1: Effects of deuterium oxide on the intensities of chemiluminescence of
penicillin G 92
Table 6-2: The responds of spraying the three reagents on sample PGKOj and sample
KO2 92
Table 6-3: The running time of TLC developments 92
Table 6-4: The retention times of the reference compounds and sample PGKO2 93
Table 6-5: The operating conditions of HPLC 94
Table 6-6: The products of recombination including dimerization after penicillin G
reacting with potassium superoxide 95
Table 6-7: The major products of hydrolysis of penicillin G after interacting with
potassium superoxide 96
viu
Table 6-8: The major products of oxidation of penicillin G after interacting with
potassium superoxide 97
Table 6-9: The six p-lactam antibiotics that emit no chemiluminescence 98
Table 6-10: The seven P-lactam antibiotics that emit chemiluminescences 99
IX
LIST OF FIGURES
Figure page
Figure 1-1: Bonding in the diatomic oxygen molecule 19
Figure 1-2: Potential energy curves for the three low-lying electronic states of
molecule oxygen 20
Figure 1-3: State correlation diagrams for the reactions of the three low-lying states
of molecule oxygen with a diene to produce endoperoxide in triplet (T) and
singlet (S) states 21
Figure 1-4: Possible mechanism for formation of oxygen free radical during ischemic
reperfusion 22
Figure 1-5: Parent penicillin 23
Figure 1-6: Parent cephalosporins 24
Figure 1-7: Oral beta-lactam antibiotics 25
Figure 1-8: Nonclassical p-lactam antibiotics 26
Figure 1-9: Reaction scheme of luminol based chemiluminescence detection 27
Figure 3-1: Schematic diagram of setup for chemiluminescent measurement in static
mode 46
Figure 4-1: The chemiluminescence of luminol following the oxidation by (a) HjOj
and(b)K02 51
Figure 4-2: The chemiluminescence of luminol reacting with KOj in CH3OH. (a) at
starting time, (b) two hours later, (c) three hours later 52
Figure 4-3: The correlation between time and the chemiluminescent intensities of
luminol after repeating the injections of 50 ^1 of 1 :1 CH3CN-CH3OH solution
containing o.l M KO2 into 2.5 ml of 10"' M luminol every 15 minutes 53
Figure 5-1: The comparison of chemiluminescent intensities of thirteen P-lactam
antibiotics following the oxidation by KOj 58
Figure 5-2: The calibration curve of penicillin G 59
Figure 5-3: The calibration curve of dicloxacillin 60
Figure 5-4: The photocounting chemiluminescent spectrum (intensity vs. wavelength)
of penicillin G reacting with superoxide 61
Figure 5-5: The photocounting chemiluminescent spectrum (intensity vs. wavelength)
of luminol reacting with hydrogen peroxide 62
Figure 5-6: The typical chemiluminescent spectrum (intensity vs. time) of P-lactam
antibioticals 63
Figure 5-7: The chemiluminescent spectrum (intensity vs. time) of ampicillin 64
Figure 6-1: The profile of chemiluminescences of penicillin G in different solutions
with varied deuterium oxide to water ratios 100
Figure 6-2:'H-NMR spectrum of potassium penicillin G in DjO 101
Figure 6-3:'^C-NMR spectrum of potassium penicillin G in DjO 102
Figure 6-4: APT spectrum of potassium penicillin G in DjO 103
Figure 6-5:'H-NMR spectrum of the degradation products of potassium
penicillin G in D2O after the oxidation by solid KO2 104
Figure 6-6: '^C-NMR spectrum of the degradation products of potassium
penicillin G in D2O after the oxidation by solid KOj 105
Figure 6-7: 'H-NMR spectrum of potassium penicillin V in D2O 106
Figure 6-8: '^C-NMR spectrum of potassium penicillin V in D2O 107
Figure 6-9: APT spectrum of potassium penicillin V in DjO 108
Figure 6-10: 'H-NMR spectrum of the degradation products of potassium
penicillin V in D2O after the oxidation by KO2 109
Figure 6-11: '^C-NMR spectrum of the degradation products of potassium
penicillin V in DjO after the oxidation by KO2 110
XI
Figure 6-12: The schematic diagrams of TLC assays on sample PG and sample
PGKO2 Ill
Figure 6-13: the schematic diagrams of preparing the samples for mass
spectroscopy by the TLC separations 112
Figure 6-14: The FAB-mass spectrum of the sample from spot 13 113
Figure 6-15: The chromatograms of sample PG (a) and sample PGKO2 (b). The
buffer system: pH5.66 phosphate buffer containing 0.1% acetonitrile 114
Figure 6-16: Schematic of major processes occurring in electrospray 115
Figure 6-17: The total chromatograms of sample PG and sample PGKO2 116
Figiu-e 6-18: The zoom-in chromatogram of the new oxidation products
generated by sample PGKO2 117
Figure 6-19: The scheme of generation of sulfoxides after penicillin G reacts with
potassium superoxide 118
Figure 6-20: The proposed chemiluminescent mechanism of penicillin G after
reacting with potassium superoxide 1 19
Figure A-1: The positive HPLC/ESI-MS/MS ofbenzylpenilloic acid 125
Figure A-2: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic
acid 126
Figure A-3: The negative HPLC/ESI-MS/MS ofbenzylpenilloic acid 127
Figure A-4: The interpretation of the negative HPLC/ESI-MS/MS ofbenzylpenilloic
acid 128
Figure A-5: The positive HPLC/ESI-MS/MS of penicillin G and MS/MS of m/z 335. .129
Figure A-6: The negative of HPLC/ESI-MS/MS of penicillin G and MS/MS of
m/z 333 130
Figure A-7: The interpretations of the positive and negative HPLC/ESI-MS/MS of
penicillin G 131
^iii*=^
Figure A-8: The positive HPLC/ESI-MS/MS of benzylpenillic acid 132
Figure A-9: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilhc
acid 133
Figure A-10: The positive HPLC/ESI-MS/MS of benzylpenicillenic acid 134
Figure A-1 1 : The interpretation of the positive HPLC/ESI-MS/MS of
benzylpenicillenic acid 135
Figure A-12: The negative HPLC/ESI-MS/MS of benzylpenicillenic acid 136
Figure A-1 3: The interpretation of the negative HPLC/ESI-MS/MS of
benzylpenicillenic acid 137
Figure A-14: The positive HPLC/ESI-MS/MS of benzylpenicilloic acid 138
Figure A-1 5: The interpretation of the positive HPLC/ESI-MS/MS of
benzylpenicilloic acid 139
Figure A-16: The negative HPLC/ESI-MS/MS of benzylpenicilloic acid 140
Figure A-1 7: The interpretation of the negative HPLC/ESI-MS/MS of
benzylpenicilloic acid 141
Figure B-1: The positive HPLC/ESI-MS/MS of the sulfoxide of penicillamine
dimer 143
Figure B-2: The interpretation of the positive HPLC/ESI-MS/MS of the sulfoxide
of penicillamine dimer 144
Figure B-3: The negative HPLC/ESI-MS/MS of the sulfoxide of penicillamine
dimer 145
Figure B-4: The interpretation of the negative HPLC/ESI-MS/MS of the sulfoxide
of penicillamine dimer 146
Figure B-5: The positive HPLC/ESI-MS/MS of benzylpenilloic acid sulfoxide 147
Figure B-6: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic
acid sulfoxide 148
xui
Figure B-7: The negative HPLC/ESI-MS/MS of benzylpenilloic acid sulfoxide 149
Figure B-8: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilloic
acid sulfoxide 150
Figure B-9: The positive HPLC/ESI-MS/MS of benzylpeniUic acid sulfoxide 151
Figure B-10: The interpretation of the positive HPLC/ESI-MS/MS of benzylpeniUic
acid sulfoxide 152
Figure B-11: The negative HPLC/ESI-MS/MS of benzylpeniUic acid sulfoxide 153
Figure B-12: The interpretation of the negative HPLC/ESI-MS/MS of benzylpeniUic
acid sulfoxide 154
Figure B-13: The positive HPLC/ESI-MS/MS of benzylpenicillenic acid sulfoxide 155
Figure B-14: The interpretation of the positive HPLC/ESI-MS/MS of
benzylpenicillenic acid sulfoxide 156
Figure B-15: The negative HPLC/ESI-MS/MS of benzylpenicillenic acid sulfoxide 157
Figure B-16: The interpretation of negative HPLC/ESI-MS/MS of
benzylpenicillenic acid sulfoxide 158
Figure B-17: The positive HPLC/ESI-MS/MS of penicillin G sulfoxide 159
Figure B-18: The interpretation of the positive HPLC/ESI-MS/MS of penicillin G
sulfoxide 160
Figure B-19: The negative HPLC/ESI-MS/MS of penicillin G sulfoxide 161
Figure B-20: The interpretation of the negative HPLC/ESI-MS/MS of penicillin G
sulfoxide 162
Figure B-21: The positive HPLC/ESI-MS/MS of compound 366a-penicillin G
sulfone 163
Figure B-22: The interpretation of the positive HPLC/ESI-MS/MS of compound
366a-penicillin G sulfone 164
XIV {' ,
Figure B-23: The positive HPLC/ESI-MS/MS of compound 366b-isomer of
penicillin G sulfone 165
Figure B-24: The interpretation of the positive HPLC/ESI-MS/MS of compound
366b-isomer of penicillin G sulfone 166
Figure B-25: The positive HPLC/ESI-MS/MS of compound 366c-isomer of
penicillin G sulfone 167
Figure B-26: The interpretation of the positive HPLC/ESI-MS/MS of compound
366c-isomer of penicillin G sulfone 168
Figure B-27: The negative HPLC/ESI-MS/MS of compound 366c-isomer of
penicillin G sulfone 169
Figure B-28: The interpretation of the negative HPLC/ESI-MS/MS of compound
366c-isomer of penicillin G sulfone 170
Figure B-29: The positive HPLC/ESI-MS/MS of benzylpenicilloic acid sulfoxide 171
Figure B-30: The interpretation of the positive HPLC/ESI-MS/MS of
benzylpenicilloic acid sulfoxide 172
Figure C-1: The positive HPLC/ESI-MS/MS of penicillamine dimer 174
Figiire C-2: The interpretation of the positive HPLC/ESI-MS/MS of penicillamine
dimer 175
Figure C-3: The positive HPLC/ESI-MS/MS of compound 440 176
Figure C-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 440.. ..177
Figure C-5: The positive HPLC/ESI-MS/MS of compound 632 178
Figure C-6: The interpretation of the positive HPLC/ESI-MS/MS of compound 632.. ..179
Figure C-7: The negative HPLC/ESI-MS/MS of compound 632 180
Figure C-8: The interpretation of the negative HPLC/ESI-MS/MS of compound 632. ..181
Figure C-9: The positive HPLC/ESI-MS/MS of compound 642 182
XV
Figure C-10: The interpretation of the positive HPLC/ESI-MS/MS of compound
642 183
Figure C-11: The negative HPLC/ESI-MS/MS of compound 642 184
Figure C-12: The interpretation of the negative HPLC/ESI-MS/MS of compound
642 185
Figure D-1: The negative HPLC/APCI-MS of benzoic acid 187
Figure D-2: The interpretation of the negative HPLC/APCI-MS of benzoic acid 188
Figure D-3: The positive and negative HPLC/ESI-MS/MS of compound 336 189
Figure D-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 336.... 190
Figure D-5: The interpretation of the negative HPLC/ESI-MS/MS of compound 336.. .191
Figure D-6: The positive HPLC/ESI-MS/MS of compound 511 192
Figure D-7: The interpretation of the positive HPLC/ESI-MS/MS of compound 51 1.... 193
Figure E-1: The negative HPLC/ESI-MS/MS of compound 269 195
Figure E-2: The interpretation of the negative HPLC/ESI-MS/MS of compound 269.... 196
Figure E-3: The positive HPLC/ESI-MS/MS of compound 353 197
Figure E-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 353. ...198
xvi
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfilhnent of the
Requirements for the Degree of Doctor of Philosophy
THE CHEMILUMINESCENCES AND DEGRADATIONS OF p - LACTAM
ANTIBIOTICS AFTER OXIDATION BY POTASSIUM SUPEROXIDE
By
Jingshun Sun
August 1998
Chairman: John H. Perrin, PhD ' ,
Major Department: Medicinal Chemistry
Penicillin G is investigated as a model to study how thirteen P - lactam antibiotics
emit chemiluminescences with different intensities after oxidation with potassium
superoxide. The degradation products of aqueous Penicillin G, which reacts with
potassium superoxide, are analyzed via direct infusion by HPLC - electrospray ionization
(ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry. A
number of products derived from the hydrolysis, oxidation, polymerization and
chemiluminescent reaction are identified, and reaction schemes are proposed.
A positive correlation is observed between the chemiluminescences and the
autoxidations initiated by the singlet oxygen molecule-OjCAg). Deuteration experiments
xvii
reveal that the intensity of the chemiluminescence from Penicillin G in deuterium oxide is
approximately ten times as strong as that in neutral water. It is consistent with the longer
lifetime of singlet oxygen molecule in deuterium oxide. The results presented here
reinforce that singlet oxygen molecules play a crucial role in structural breakage of
protein or DNA as well as in the specific disease entities.
xvui
CHAPTER 1
INTRODUCTION
Reactive Oxygen Species
A free radical can be defined, as any atom or molecule possessing one or more
unpaired electrons. It can be anionic, cationic, or neutral. In biological and other related
fields, the major free radical species of interest have been oxygen free radicals. The term
oxygen free radicals includes the superoxide anion radical (O2" ), the hydroperoxyl radical
(HO2 ), the hydroxyl radical (OH ), and the peroxide radical (ROO , R = Lipid). Actually
these free radicals belong to a group of oxygen molecules called reactive oxygen species
because they have sfronger oxidizing ability than oxygen itself Besides the oxygen free
radicals these reactive oxygen species consist of hydrogen peroxide (HjOj), hypochlorous
acid (HOCl), lipid peroxide (LOOH), and singlet oxygen ('Oj).
Singlet Oxvgen
When the configuration of the oxygen molecule orbital is described as
KK(2ag)^(2aJ^(3ag)^(l7:u)''(l7ig)'', the ground state oxygen itself is a biradical, as shown in
Figure 1-1. This triplet oxygen has two unpaired elecfrons, each located in a different n*
antibonding orbital with the same spin quantum number. In accordance with Pauli's
principle that a pair of electrons in an atomic of molecular orbital would have antiparallel
,.• '
js-t.^
spins, this imposes a restriction on the oxidation by the ground state oxygens because the
new electrons removed concomitantly from nonradical molecules must be of parallel spin
so as to fit into the vacant spaces in the n* orbitals. The transition metals found at the
active site of oxidase and oxygenase enzymes have shown the capability of accepting or
donating one electron at a time to increase the reactivity of ground state oxygen and to
overcome the spin restriction. Another way of enhancing the reactivity of ground state
oxygen is to change the spin direction of one of the two electrons in the n* orbital. Two
higher energy singlet states of oxygen molecule were found by Childe and Mecke [1] (the
'Eg* in 1931) and by Herzberg [2] (the 'Ag in 1934). These two forms of the oxygen
molecule had an excess energy fraction of a chemical bond, 37.5 and 22.5 Kcal mol"',
respectively, as shown in Figure 1-2.
Singlet OzCSg*) is readily relaxed to 02('Ag) before it has time to react with
anything. By definition, delta singlet oxygen is not a free radical. Like oxygen free
radicals, however, if singlet oxygen is released in biologic systems, it is capable of
rapidly oxidizing many molecules. In vitro chemical studies of singlet oxygen show
singlet oxygen has the same damaging effects as does the superoxide anion toward
various biological structures including nucleic acids, proteins, and lipids [3].
The two principle reactions characteristic of singlet oxygen are cycloaddition and
the 'ene' reaction [reaction (1) and (2), respectively]:
I 4 \/'^\/
y — K r c^=c R
R R
= + R C C^ R. C
/ \
R R
R
The formation of endoperoxides shown in reaction (1) requires a conjugated
double bond. The formation of hydroperoxides as shown in reaction (2) requires an allyhc
hydrogen. When the double bonds are surrounded by electron-donating groups, the
activation energy for both reactions can be reduced to nearly zero and the rate constant
then approaches its maximum value of about 10^ 1 mol"' s"' [4]. The formation of
dioxetanes in singlet oxygen reactions appears to have a much higher activation energy
and is observed chiefly when other pathways are blocked [5,6]. This is consistent with the
predictions of the Woodward-Hoffman selection rules for concerted cycloaddition
reactions [7]. Such orbital symmetry considerations and more quantitative calculations
indicate that the 'Ag* component is the only one which correlates directly with the ground-
state products in reaction (1) [7]. These correlations are illustrated by the potential energy
curves shown in Figure 1-3.
Superoxide Anion
Given a single electron the ground state oxygen molecule becomes a superoxide
anion (Figure 1-1). It is formed in almost all aerobic cells [8,9,10]. Superoxide chemistry
differs greatly regarding whether reactions are carried out in aqueous solution or in
organic solvents. In nonpolar envirormients superoxide is a powerful base, nucleophile,
and reducing agent [11,12]. Superoxide can also act as an oxidizing agent, but this ability
is only seen with compounds that can donate protons. In aqueous solution, superoxide
undergoes the so-called dismutation reaction to form hydrogen peroxide and oxygen. The
overall reaction can be written as
202- + 2H^ ^ H2O2 + O2 (3)
Under physiological conditions, even though uncatalyzed, this reaction can be very fast,
i.e. 10^ M"' S"' [13]. The reactivity of superoxide is greatly reduced in water, and
dismutation reaction being favored in this media.
The early superoxide theory of oxygen toxicity had been established on the
accumulation of evidence showing that superoxide dismutation (SOD) enzymes, which
remove superoxide by accelerating the dismutation reaction, are of great importance in
allowing organisms to survive in the presence of oxygen and to tolerate the increased
oxygen concentrations [8-10, 14, 15]. Since SOD enzymes are specific for superoxide as
the substrate, it follows that superoxide must be a toxic species. Indeed, many damaging
effects summarizing in Table 1-1 are associated with superoxide generating systems [16].
However, the damage effects listed in Table 1-1 were produced by superoxide in aqueous
solution, moreover, superoxide has proven to be relatively unreactive toward most
biological components [17]. Therefore, it seems unlikely that superoxide alone can create
such damage.
Historically, it is generally accepted that the superoxide anion radical is not a
particularly reactive species but is potentially toxic. It can be transformed into the highly
dangerous hydroxyl radical following the metal catalyzed Haber- Weiss reaction [8].
TT ^ ^ transition metal ^ ^^, ^^^
«^02 + O.- eatalyst ^ ^^ + ^^' + ^^ (4)
The hydroxyl radical will react with a wide range of biological molecules in its vicinity.
Inspection of Table 1-1 shows that the metal-dependent Haber- Weiss reaction
does not explain all, as there are several damage examples of prevented by SOD but not
by catalase. In many other cases, for example the organism Streptococcus Sanguis, whose
growth appears to be independent of the availability of iron [18] as it contains no heme
compounds and lacks catalase and peroxidase, is damaged by exposure to a superoxide-
generating system, but this damage is not prevented by scavengers of OH. Actually, there
is no direct evidence of hydroxyl radical in any superoxide-hydrogen peroxide reacting
enzymatic system, and the Haber- Weiss proposal is totally based on the inhibitory effects
of SOD enzymes and that of hydroxyl radical scavengers. On the other hand the presence
of singlet oxygen molecule Oj ('Ag) generated in the Haber - Weiss reactions is proved
by the characteristic 1268 - nm chemiluminescence emission spectrum of single
molecule oxygen [19]. In view of the fact that singlet Oj ('Ag) has a sufficiently long
lifetime to diffuse in cells and selectively damage some cell constituents, singlet Oj ('Ag)
may be the prime oxidant.
Ironically, it is has been known since 1977 that singlet Oj ('Ag) can be produced
either in the non - enzymatic dismutation reaction of superoxide or in the metal catalyzed
Haber - Weiss reaction [20]. Singlet O2 (' Ag) has never drawn as much attention as the
hydroxyl radical does due to its low concentration and the difficulty of detection.
.. iv ' .' '• Oxygen Toxicity and Diseases
Abnormal production of reactive oxygen species have been associated with a
nimiber of diseases including rheumatoid arthritis, pulmonary emphysema caused by
cigarette smoking, ischemic - reperfusion tissue injury, carcinogenesis, aging, and
neurodegeneration disorders such as Alzheimer's Disease, Parkinson's Disease, etc.
Conclusive evidence suggests that all cellular components appear to be sensitive to the
reactive oxygen species damage, lipids, proteins and nucleic acids being the most
susceptible to this injury.
Rheumatoid Arthritis
Rheumatoid arthritis has many characteristics of a free-radical-produced disease.
In rheumatoid arthritis, the synovium of the joins is swollen with an inflammatory
infiltrate, and the joint cartilage becomes eroded. Production of synovial fluid, which
lubricates the joint, is increased, but its viscosity is decreased because of the breakdown
of the polymer hyaluronic acid, which acts as a lubricant. This breakdown may be caused
by oxygen free radicals produced by neutrophils that accumulate in large numbers in the
affected joints of patients with rheumatoid arthritis [21]. Increased levels of the products
of lipid peroxidation reactions are found both in the synovial fluid and in the plasma of
patients with active rheumatoid arthritis [22], this indicates the involvement of hydroxyl
radical although the exact cause is unknown.
Pulmonarv Emphysema
The correlation between puhnonary emphysema and a, -protease inhibitor
deficiency has led to the hypothesis that the lung connective tissue damage is
characteristically associated with smoking, and this lung tissue injury results from an
impaired ability of protease inhibitors to protect lung elastin from damage caused by
leukocyte protease, hi human, a, -pro tease inhibitor is the major serum antipro tease, and
following bronchoalveolar lavage of normal persons, it is responsible for more than 90%
of such antielastase activity [23]. Electron spin resonance specfroscopy has shown that
cigarette smoke contains a variety of oxygen- and organic- based free radicals [24] and
that these free radicals completely prevent a, -protease inhibitor activity [25]. A recent
interesting in vitro observation demonstrates that catalase and the antioxidants
glutathione and ascorbic acid completely prevent the removal of elastase inhibitory
capacity of a,-protease inhibitor by cigarette smoke.
Ischemic-Reperfiision Tissue Injury
Ischemic-reperfusion tissue injury can occur in several tissues in addition to the
heart [26, 27]-for example, the small intestine, gastric mucosa, kidney, liver, and skin.
The enzyme xanthine oxidase is widely distributed among these tissues. The intestine,
lung, and liver have particularly high levels. The intestinal mucosa is very sensitive to
ischemic-reperfusion injury. One hour of regional intestinal ischemia produces a
considerable increase in capillary permeability, an effect that is reduced by superoxide
dismutase [28]. Biochemical changes during the ischemic period are thought to be the
basis for a burst of production of firee radicals on reintroduction of molecular oxygenation
at reperfusion-Figure 1-4 [29]. As shown in Figure 1-4, during ischemia, adenosine
triphosphate (ATP) is metabolized to the substrate hypoxanthine, and the enzyme
xanthine dehydrogenase is converted to xanthine oxidase by a protease activated by
increased free calcium. On reperfusion, hypoxanthine reacts with molecule oxygen in the
presence of xanthine oxidase to form superoxide anion radicals. In the presence of iron
salts, superoxide anion radicals can form hydroxyl radicals, which can bring about a
number of damages.
Carcinogenesis
Carcinogenesis is thought to occur in two stages. In the initiation stage, a
physical, chemical, or biological agent directly causes an irreversible alternation in the
molecular structure of DNA of the cell. This alternation is followed by a promotion stage,
in which the expression of the genes that regulate cell differentiation and growth is
altered. Oxygen free radicals play a role mostly in the promotion phase of carcinogenesis
[30]. Hyperbaric oxygen, superoxide anion radical, and certain organic peroxides are
tumor promoters but may also be weak complete carcinogens [31]. In contrast, many
antioxidants are antipromoters and anticarcinogens [32]. However, the literature is not
extensive enough to fully describe how the molecular structure of DNA is broken down
by oxygen free radicals or other unknown oxygen species. Further investigations are
needed. "
Aging .^^.^
, The universality of aging implies that its cause is basically the same in all species.
A free radical hypothesis of aging has been proposed. It suggests that the free radical
produced during normal metabolism of the cell over time damages DNA and other
macromolecules and leads to degenerative diseases, malignant lesions, and eventual death
of the animal [33, 34]. Oxidative DNA damage is rapidly and effectively repaired. The
human body is continually repairing oxidized DNA. An estimated several thousand
oxidative DNA damage sites are present in the human cell every day, most of which are
repaired [35]. A small fraction of unrepaired lesions could cause permanent changes in
DNA and might be a major contributor to aging and cancer. The hypothesis that oxygen
radicals play a role in aging is supported by the observation that, in general, long-lived
species produce fewer endogenous free radicals because of their lower metabolic rate
[36]. Long-lived animals also have more superoxide dismutase than do their short-lived
counterparts, and animal species with the longest life-spans have the highest levels of
10
superoxide dismutase [37]. A consequence of the free radical hypothesis of aging is the
concept that free radical scavenging agents might be used to prevent aging. Several
antioxidants, including vitamin E [38] and butylated hydroxytoluene (BHT) [39] have
been tested in animals and have yielded equivocal results. Interestingly, a study of a self-
selected group of high dose vitamin E users who were 65 years or older showed an
increased mortality associated with consumption of more than 1,000 lU of vitamin E a
day [40]. The correlation between life expectancy, life-span, and dietary antioxidant
intake in humans remains to be demonstrated.
Neurodegenerative Disorders .
The central nervous system (CNS) is particularly vulnerable to free radical
damage because of the following anatomical, physiological and biochemical reasons [41]:
1. Relative to its size, there is an increased rate of oxidative metabolic activity.
2. There are relatively low levels of antioxidants (e.g., glutathione) and
protective enzyme activity (e.g., glutathione peroxidase, catalase, superoxide
dismutase).
3. Abundant readily oxidizable membrane polyunsaturated fatty acids are
present.
4. Endogeneous generation of reactive oxygen species via several specific
neurochemical reactions are possible.
5. The CNS contains non-replacing neuronal cells, once damaged they may be
dysfiinctional for life.
11
6. The CNS neural network is readily disrupted.
The evidence for the role of free radicals in CNS disorders is generally indirect
due to their extremely reactivity, as well as due to the general inaccessibility of the brain.
In vivo, direct biochemical monitoring is impossible, however, numerous experiments
still indicate the association between free radicals and several major CNS disorders as
shown in Table 1-2.
Chemotherapy
The therapies based on modulation of the formation of free radicals include:
Removal of Free Radicals bv Superoxide Dismutase and Vitamin E
Precedents have been made to the possible protection by superoxide dismutase
against some kinds of free radical damage in animals. Orgotein, a bovine Cu-Zn
superoxide dismutase, has been used as an anti-inflammatory protein drug in veterinary
practice [60]. This agent is reported to have beneficial effects in treating rheumatoid
arthritis, Duchenne's muscular dystrophy, and radiation-induced cystitis [61, 62, 63], but
fiirther double-blind placebo controlled studies are needed to confirm these findings [64].
Human recombinant superoxide dismutase has recently been produced, and trials are
planned to evaluate its therapeutic efficacy in myocardial infraction, kidney
fransplantation, and bronchopulmonary dysplasia in premature infants [65].
Vitamin E had been shovra by studies with isolated cells and animals to protect
against damage caused by free radicals [66]. Under normal conditions, radical scavenging
12
by vitamin E is just one of many mechanisms involved in minimizing jfree radical-related
tissue damage. Many of the numerous clinical studies involving large doses of vitamin E
have not been adequately controlled, and the therapeutic benefits of vitamin E remain
controversial [67].
Increasing Formation of Free Radicals
The main emphasis in treating human disease caused by free radicals is to
decrease the formation of free radicals or limit their reaction at critical sites within the
body. However, the formation of free radicals might be considered as a useftil therapeutic
strategy when the desired therapeutic effect is to damage or kill certain cells, but
selectivity for the desired site is a priority. The various ions plus short-lived and reactive
free radicals formed from the interaction of ionizing radiation and water have shown
cell-killing effect [68]. Two major free radicals, hydroxyl radical and the aquated electron
(e'«,), result from the radiolysis of water. The hydrated elecfron reacts by neucleophilic
addition to produce radical anions, it further reacts with molecular oxygen to produce
organic peroxide radicals:
R + eaq ^ R (5)
R' + O^ ^ RO2 (6)
A surprisingly large number of anticancer drugs in use today, almost half of those
approved drugs in the United States, can form free radicals. This fact, considered together
with the observation that tumor cells may be deficient in the same enzymes, i.e.
13
superoxide dismutase and catalase, that normally protect cells from free radical damage,
has led to suggestions that free radicals might be involved in the antitumor activity of
some of these drugs [69]. The best evidence of a free radical involvement in the cytotoxic
effect of an anticancer drug is from the glycopeptide antibiotic bleomycin [70].
Bleomycin binds to DNA and in the presence of ferrous iron and oxygen cleaves DNA. A
free radical species, possibly the hydroxyl radical is formed in near vicinity to the DNA
and results in its degradation [71].
During the past three decades, the classic fransition metal catalyzed Haber- Weiss
reaction has met with almost universal acceptance and forms the foundation of this very
active research field. Although the interpretation of evidence presented on this subject has
been difficult because of the problems in identifying the free radicals, the toxicity of
hydroxyl radicals generated by the Haber- Weiss reaction have been blamed for almost
every disease caused by reactive oxygen species, and the chemotherapy is exclusively
based on modulating the formation of free radicals. That the oxygen free radicals might
be involved in human disease is not surprising in view of their existence in many
biological systems, which have seemingly evolved elaborate protective mechanisms
which normally effectively prevent damage by these radicals. In certain circumstances
singlet oxygen has been shown to create similar damages to the hydroxyl radical. Few but
sfrong evidences already support the argument that in some diseases it is singlet oxygen,
not the hydroxyl radical which acts as a prime oxidant. This controversy needs to be
14
clarified in order to better understand the oxygen toxicity and to invent new therapeutic
strategy as well as new antioxidants.
Chemiluminescence and p-Lactam Antibiotics
P-Lactam Antibiotics
The P-Lactam structure of penicillin was first proposed by Abraham and Chain in
1943, 14 years after Fleming made his first observations on the antibacterial action of
penicillin on a plate seeded with staphylococci. It was opposed by those committed to an
alternative thiazolidine-oxazolone structure, however, the P-lactam structure was finally
established beyond doubt in 1945 when an X-ray crystallographic analysis by Dorothy
Hodgkin and Barbara Low came to a successfiil conclusion[72]. Penicillin was
pharmacologically ahead of its time. In the early 1940s, as penicillin research was just
getting underway in earnest, the sulfa drugs were a revolutionary new concept in
chemotherapy. The discovery of sulfa drugs, which would selectively react with bacteria,
was a major innovation in chemotherapy [73]. By 1948 the outstanding value in medicine
of benzylpenicillin had been firmly demonstrated, more and more scientists were
encouraged to develop new P-lactam antibiotics. Chemical modifications of P-lactam ring
and the introduction of specific side chains have led to a new series of compounds with
characteristic biological activities. Due to endless efforts, a large family of P-lactam
antibiotics is established and still grows rapidly. Representative structures of those drugs
are shown in Table 1-3. Many P-lactam antibiotics can be classified into four classes
IS
according to their clinical usefulness and pharmacokinetic properties [74]: (I) parental
penicillins (Figure 1-5), (II) parental cephalosporins (Figure 1-6), (III) oral penicillins and
cephalosporin (Figure 1-7), (IV) nonclassical p-lactam antibiotics (Figure 1-8). The
analytical means for particular P-lactam antibiotics are different because of their diverse
chemical, physical, and biological properties. In the case of the analysis of penicillin G,
many methods are adopted [75] including titration, colorimetry and UV
spectrophotometry, florescence, HPLC, thin layer chromatography, gas-liquid
chromatography, electrophoresis, polarography, enzyme electrodes, isotopic assay,
biological-based microbiological assay, immunoassay, and enzyme-aided assay
(hydrolysis). In 1991, Schulman and Perrin revealed that cephalothin has the ability to
prolong and intensify the chemiluminescence derived from the cobalt (Il)-luminol-
hydrogen peroxide system. It can be used as the basis for the determination of
cephalothin in the range of 0.4-400 ^ig ml"'[76]. Post column chemiluminescent detection
after liquid chromatography has also been used to prevent mixing problems and so
increase the reproductivity and sensitivity.
Chemiluminescence
Chemiluminescence can be defined as the emission of light as a result of the
generation of electronically excited states formed in a chemical reaction. The range of
wavelengths of light emitted is suprisingly large-from the near ultravilet to the infra-red.
Chemiluminescence is "cold light." The energy is released in the form of light rather than
16
heat. It has wider variety of color than the bioluminescence of the firefly but is far less
efficient. Analysis by chemiluminescence has simple instrumentation, has excellent
sensitivity, has very low limits of detection, and has great selectivity and wide dynamic
ranges. The efficiency of a luminescent reaction is defined as the number of photons
emitted per reacting molecule. The quantum yield is usually written as:
cDcl = ^rxOesX<1^f (7)
where Or is the yield of product, O^s the number of molecules entering the
excited state and Op is the fluorescence quantum yield. High yields of excitation
without strong visible emission are possible if Op is very low, as is the case for simple
dioxetanes. Phosphorescence is difficult to observe under the conditions of most of the
reactions, but in principle Op^,,, can replace Op . Sensitized chemiluminescence results
when transfer of energy takes place between chemiluluminescent reactants and
fluorescent acceptors. The efficiency of this energy transfer (ET) must be taken into
account:
^cl = <I5rX^esX<I^etX^p (8)
where Op, is the fluorescence efficiency of the acceptor, and O^x is the efficiency of
energy transfer. Addition of fluorescent acceptors can enhance light emission in the case
of dioxetanes. The analytically useful chemiluminescent emitters are listed in Table 1-4.
If a target analyte can be determined via HPLC chemiluminescence then it
probably has one of three characteristics:
17
1 . it either emits chemiluminescence when mixed with a specific reagent;
2. it catalyzes chemiluminescence between other reagents;
3. it is suppresses chemiluminescence between other reagents.
Since not many pharmaceuticals can give out chemiluminescence, most analysis of
HPLC chemiluminescence are based on 2) and 3) reactions, in which luminol (5-amino-2,
3-dihydro-l,4-phthalazinedione), reacts with oxidants like hydrogen peroxide in the
presence of a base and a metal catalyst to produce an excited state product (3-
aminophthalate, 3-APA) which gives off light at approximately 425 nm. When a
compound eluting from the LC column enhances or suppresses this background light, the
amount of light increased or decreased will represent the amount of analyte. The reaction
system is described in Figure 1-9. Recently Garcia Campana, Baeyens, and Zhao
reviewed chemiluminescence detection in capillary electrophoresis [92]. The combination
of capillary electrophoresis, which is versatile and robust, and chemiluminescence-based
reactions, which are extremely sensitive, is promising for numerous applications in fields
such as environmental analysis and medicine. However, it is still not used as a routine
technique. Several problems need to be solved, for instance, chemiluminescence lacks
selectivity, the emissions of chemiluminescences depend on environmental factors; and
the optimization of the volume and geometry of the detection cell and connecting
hardware.
18
In our experiments the investigation of the behavior of the reactive oxygen
species has always been the priority. Eleven p-lactam antibiotics have been reacted with
potassium superoxide and the oxidation as well as hydrolysis of penicillin G have been
examined. The deuteration experiment using penicillin G reinforces the concept that
singlet oxygen is generated by superoxide dismutation and that singlet oxygen play an
important role in tissue injury and related diseases.
' - * i.
19
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Figure 1-2: Potential energy curves for the three low-lying electronic states of molecular
oxygen.
21
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Figure 1-3: State correlation diagrams for the reactions of the three low-lying states of
molecule oxygen with a diene to produce endoperoxide in triplet (T) and singlet (S)
states.
22
Krhpmlfl Adenosine
iscnemia •, Xanthine
Inosine dehydrogenase
Hypoxanthine
Reperfusion
Protease
Xanthine
H202<
I Iron salts
HO' + HO -+ O2
Figure 1-4: Possible mechanism for formation of oxygen free radical during ischemic
reperfusion.
23
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Nonclassical beta-lactam antibiotics
Nocardicin A
Clavulanic acid
CP-45899
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Thienamycin
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N-acetylthienamycin
MK-0787
Carpet imycin
MM-4550
PS-4-7
Figure 1-8: Nonclassical P-lactam antibiotics.
In Figure 5, 6, 7, 8 the compounds with similar structures are drawn in the same block.
27
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30
Table 1-3: Family of P-lactam antibiotics.
Parental
Penicillin
Parental
Cepholosporin
Nonclassical
Structure
R
HO
K
CHj
CH,
Penicillin G, R= <f ^CHj-
Cephalothin, R,= ^ /^-CHj-
R2=-OCOCH3
pH
HO — N,
HjN
Nocardicin A
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Clavulanic Acid Sulfones
Thienamycin
31
Table 1-4: Analytical useful chemiluminescent emitters.
Chemiluminescent
Emitters
^max (wavelength Region)
Reference
3-Aminophthalate (from
Luminol)
425 nm
77
CHaSe
750-825 nm
78
CN
383-388 nm
79
HCF
475-750 nm
80
HCHO
350-500 nm
81
HF
670-700 nm
80
HSO
360-380 nm
82
IF
450-800 nm
83
N-methylacridone (from
lucigene)
420-500 nm
84
Na
589 nm
85
NO2
1200 nm
86
OH
306 nm
82
Oxyluciferin (from
luciferin)
562 nm
87
Ru(bpy)3
600 nm
88
S2
275-425 nm
89
SF2
550-875 nm
90
SO2
260-480 nm
91
CHAPTER 2
OBJECTIVES
Investigation of the chemiluminescence following the oxidation of p-lactam
antibiotics by potassium superoxide will facilitate a better understanding of the
involvement of 'Oj in numerous human diseases caused by oxygen toxicity.
Recently more and more people began to realize that the generation of OH by
transition metal-catalyzed Haber- Weiss reaction is not enough to account for all the
damaging effects in the biological components. It is believed that the Haber- Weiss
reaction does not occur in biological tissues under normal conditions, but its occurrence
during pathological states, e.g. ischaemia, is possible. Even so the Haber- Weiss reaction
faces the competition from the Fenton type reaction to produce OH. In the meantime, the
existence of 'Ozis indicated in the superoxide-generation systems. Mao [93] et al. utilized
2, 2, 6, 6-tetramethyl-4-piperdone as a spin trap of electron spin resonance (ESR)
spectroscopy to study the generation of 'Oj They observed the 'Oj spin adduct signal in
the incubation of xanthine, xanthine oxidase and HjOj, and the depletion of each of the
incubation components led to a sharp decrease in 'Oj generation. 'Oj scavengers like
sodium azide inhibited 'Oj generation while the OH scavenger, ethanol, only slightly
decrease the signal intensity. Catalase inhibited 'O2 generation and H^Oj enhanced it.
32
33
They believe superoxide is capable of generating 'O2 upon reaction with HjOj, i.e. the
Haber-Weiss reaction. Interestingly they also found that the decomposition of KO2
generated 'O2, which is consistent with the experimental results obtained by Khan et al. in
1970 [94], 1976 [95], 1981 [96], and 1987 [97]. However, Nilsson and Keams [98] in
1974 challenged this conclusion with a deuteration study. They adopted the same reacting
system as did Khan and injected DjO rather than HjO into dry dimethyl sulfoxide
(DMSO) containing KOj, expecting increased intensity of chemiluminescence from the
relaxation of OzC'Ag) to ^Oz because of the large differences in lifetimes in the two
solvents: 2 jxsec in H2O vs. 20 |xsec in DjO. However, no enhancement of
chemiluminescent emission was observed, and no oxidation of the "Oj-acceptor
tetramethylene (TME) could be detected in this system. Their data suggested that the
superoxide anion radical can not be a direct precursor of 'O2. Since no later evidence has
been presented, and the deuteration experiment is a very reliable approach to prove the
existence of 'O2, the Nilsson and Keams' study still casts doubt over the possibility of the
generation of 'O2 from superoxide.
Indeed, despite a tremendous amount of work, this remains one of the most
controversial, confusing, and frustrating areas of research, and the indirect detection of
OH or other reactive oxygen species based on the scavenger's effects or on the spin
trap's signals is one of the major reasons. This is because OH and 'O2 are very reactive
species, and thus are able to react with many compounds. Therefore, certain compounds
can be spin traps for both of them, and some scavengers of OH may also be effective for
34
'Oj. For example, histidine can eliminate 'Oj in addition to the molecular OH radical
[99].
In the current study based on some similar molecular structures, P-lactam
antibiotics are used as models to study how the superoxide-generating system damages
DNA molecular and protein structures. The following approaches are made to improve
the understanding of oxygen toxicity.
1). Evaluate KOj's capability as an oxidizer for chemiluminescence. The same
amount of KOj and H2O2 will be used to react with luminol respectively, and under
identical experimental conditions to check which gives the stronger chemiluminescence.
2). Study the chemical properties of KO2 in the liquid phase. A solvent must be
found to solubilize KOj and allow it to keep its activity as long as possible, because the
biological nature of superoxide anion has to be mimicked and handling solid KO2 is very
dangerous. - /<
3). Investigate whether KO2 can oxidize p-lactam antibiotics with the resultant
emissions of chemiluminescence. This will answer the question that how and why oxygen
toxicity can damage cell components.
4). Design and carry out new DjO experiments to clarify the historical
controversy.
5). Study the mechanism of chemiluminescence of P-lactam antibiotics. A
prediction of chemiluminescences from other compounds is desired.
35
6). Identify the degradation products and pathways to further confirm the
chemiluminescent mechanism and oxygen toxicity.
7). Try to answer the question: which plays the major role in structural breakage
of protein and DNA, hydroxy 1 radical or singlet oxygen?
CHAPTER 3
CHEMILUMINESCENT METHODOLOGY OF STATIC MODE
Materials and Apparatus
The chemiluminescent studies were conducted with a FL-750 Spectrofluorescence
Detector (Mcpherson, Acton, MA) in the static mode. The data was collected either by a
Servogor 120 flatbed recorder (Norma Goerz Instruments, Elk Grove Village, IL) or by
an IBM personal computer XT with Spectra Calc (Galactic Industries Corporation,
Salem, NH) as the software and DT 281 1 Analog plus Digital Input / Output board (Data
Translation Inc., Malbora, MA) as the interface. The sample was weighed accurately to
0.1 mg by a XE-IOOA Electronic Balance (Denver Instrument Company, Arvada,
Colorado). A Fisher Model 10 Accumet pH meter (Fisher Scientific, Pittsburgh, PA)
measured the pH values with the sensitivity of 0.01 pH unit (or 1 mv). A Branson
ultrasonic cleaner (Branson, Shelton, Conn.) and a Coming PC-351 Hot Plate Stirrer
(Coming Inc., Coming, NY) was used to facilitate the dissolution of solutes with low
solubilities. The saturated solution of potassium superoxide in 1 8-crown-6-ether-
acetonitrile was centriftiged with a Beckman CPR Centrifuge (Beckman Instruments Inc.,
Fullerton, CA). The sources and stmctures of chemicals utilized in these experiments are
listed in Table 3-1.
36
37
The cell compartment used for the chemiluminescent detection was modified in
order to prevent light leakage from the outside of the cell. The needle of a syringe was
connected to a curved metal tube, which was immersed in the center of the cuvette. A
black rubber stopper with a small hole allowing penetration of the metal tube covered the
cuvette holder. Since it can not travel through the curved metal tube, the light does not
enter the cuvette on withdrawal of the injection needle. Also a foam box coated with dull
black paint inside and outside served as a "dark room" to cover the whole cell
department. The setup is described in Figure 3-1. This light proof system was tested by
injecting pure water into the cuvette filled with a 10* M solution of alkaline luminol.
Even when the detector was set at its most sensitive status with the High Voltage (HV) of
Photomutiplier (PMT) at 1000 volts (Gain = 10.0), Sensitivity at 0.003, and Time
Constant at 0.25, no signal was observed. After injecting 3% H^Oj into the same solution,
the photons collected by PMT was off scale. This proves that in the current system only
the light emission inside the cuvette is detected.
Experimental Methods
After the detector and the recorder have been warmed up for several minutes, 2.5
ml of the solution to be detected, was pipetted into the cuvette, and the cuvette was then
placed into the cell holder. When the cell holder was capped by the rubber stopper and the
"dark box" was sealed, the system was ready for injection. At least three injection were
made to get an average of chemiluminescent peak heights or integrated areas with
38
reasonable reproducibility; usually the Relative Standard Deviation (RSD) was less than
10 %. The cuvette was first washed three times with tap water, then three times with
deionized water, finally rinsed with acetone, and dried with pressured air. The quartz
fluorescent cuvette was fi-equently used in these experiments. The UV quartz cuvette can
also be used, but the transparent walls must face the PMT window, and no emission
filters must be installed so that the PMT collects the total photon emission fi-om the
chemiluminescent reactions. Prior to every new experiment the detecting system was
validated by a luminol-based chemiluminescent system. A 20 |xl, 3% of H2O2 was
injected into a 2.5 ml, 10"'M of alkaline luminol solution, and a strong, broad peak was
detected. The average peak height was 238.5 mv. The experiment parameters were: Gain
= 7.10 (710 volts). Sensitivity = 0.1, Time Constant = 0.25, recorder Detecting Scale =
500 mv, recorder Paper Rate = 1 cm/min. The high concentration of reactants and stable
signal made this validation very reliable.
Preparations of Stock Solutions
All chemicals and reagents were used as provided without fiirther purification.
Preparation of Luminol Solution
The 0.001 M luminol solution was prepared by the following procedures: 88.58
mg of luminol was weighed and then dissolved in a mixture of about 150 ml deionized
water and 6.25 ml of 5 N sodium hydroxide solution with stirring. This solution was
transferred into a 250 ml volumetric flask and the volume was adjusted to 250 ml. After
39
shaking the flask gently to achieve a homogeneous mixture, the stock solution was stored
in refrigerator. The high pH of this solution benefited the further dilutions as no
additional base was needed to keep luminol in alkaline media.
Preparation of Hydrogen Peroxide Solution
The 3 % H2O2 solution was prepared by pipetting 10 ml of 30 % H2O2 solution
into a 100 ml volumetric flask then adjusted to volume with deionized water.
Preparation of Saturated Potassium Superoxide Solution
The KO2 was weighed quickly and put into dry acetonitrile, then 18-crown-6-ether
was added in excess to solubilize the KOj powder. After gentle stirring, the solution was
stored overnight in the refrigerator. The acetonitrile solution, containing excess KO2 and
crown ether, was centrifuged at 2500 rpm for 10 minutes at room temperature to leave a
saturated solution. Acetonitrile was chosen as the solvent because its polarity enhanced
the solubility of ionic species, and also because it is miscible with the aqueous solutions.
Preparation of Phosphate Buffer Solutions
Based on the Henderson-Hasselbalch equation. Table 3-2 was developed to
prepare phosphate buffer solutions. To make a buffer with pH range from 3.7 to 9.2,
certain amounts of potassium phosphate monobasic and sodium phosphate dibasic were
weighed corresponding to the clost pH value in Table 3-2, and dissolved in about 450 ml
deionized water. While the solution was stirred by a hot plate stirrer until the dissolution
was completed, the pH change was monitored by a pH meter and small amounts of dilute
40
solutions of sodium hydroxide or hydrochloric acid were added to adjust the pH value to
the desired one. Then, the solution was transferred into a 500 ml volumetric flask and
made to volume. After gently shaking the solution, the pH value of a small portion of it
was measured to confirm the final pH.
All the P-lactam antibiotic solutions were made just before the detection
experiments to minimize the effects of hydrolysis, and all the stock solutions were shaken
before being utilized.
41
Table 3-1 : Chemicals and reagents utilized in static studies of chemiluminescence.
Millipore": Millipore Corp., Bedford, MA.
Aldrich^ Aldrich Chemical Company, Inc., Milwaukee, WI 53233.
Aldrich": Aldrich Chemical Company, Milwaukee, WI53201.
Fisher'': Fisher Scientific, Fair Lawn, NJ
Mallinckrodf : Mallinckrodt, Inc., Paris, Kenturkey.
Class
Chemical or Reagent
Source
Grade
Water
Milli-Q , Ultra-pure water
system. Millipore^
Deionized
Deuterium Oxide
Aldrich''
99.9 atom % D
Methanol
Fisher
HPLC grade
Solvent
Reagent Alcohol
Fisher^
HPLC grade
n-Butanol
Fisher
Certified A. C. S.
Acetone
Fisher^
Pesticide grade
Methylene Chloride
Fisher''
Certified A. C. S.
18-Crown-6-Ether
Aldrich'
99%
Acid and
Base
85 % Phosphoric Acid
Fisher
Certified A. C. S.
Hydrochloric Acid (2N)
Fisher''
Certified
Sodium Hydroxide (5N)
Fisher"
Certified
Potassium Phosphate Monobasic
Fisher''
Primary standard
Sodium Phosphate Dibasic
Fisher''
Certified A. C. S.
Sodium Phosphate Tribasic
Mallinckrodt'
Analytical Reagent
Buffer
Buffer Solution Concentrate pH4.00
Fisher''
Certified
Buffer Solution Concentrate pH6.00
Fisher"
Certified
Buffer Solution Concentrate pH7.00
Fisher"
Certified
Buffer Solution pH 10.00
Fisher"
Certified
Oxidant
30 % Hydrogen Peroxide
Fisher"
Certified A. C. S.
Potassium Suproxide
Aldrich"
N/A (Powder)
42
Table 3-2: Chemical structures and sources of P-lactam antibiotics examined.
Beta-Lactam
Antibiotic
Stucture
Source
Azetidinone
Sulbactam
Clavulanic Acid
Cephalothin
HN — I
P
j/
V
OH
Cefotaxime
Aldrich"
Fluka'
Beecham''
LiUy
Sigma'
Table 3-2~continued
43
Beta-Lactam
Antibiotic
Structure
Source
Penicillin G
OH
Fluka"
Penicillin V
"^^
Sigma''
Ampicillin
o_/NH
OH
Interchem*^
Amoxicillin
OH
Sigma'
Piperacillin
HN-^X^.H
Sigma'
44
;-pA
Table 3-2 — continued
Beta-Lactam
Antibiotic
Structure
Source
Dicloxacillin
Sigma'
Methicillin
Sigma'
Hetacillin
OH
Beecham'
Aidrich": Aldrich Chemical Co., Inc. Milwaukee, WI.
Fluka*": Fluka Chemical Corp. Milwaukee, Wl
Beecham'; Smithkline Beecham Pharmaceuticals, Philadelphia, PA.
Lilly*: Eli Lilly and Company, Indiapolis, Ind.
Sigma': Sigma Chemical Co., St. Louis, MO.
Interchem': Interchem Corporation, Paramus, NJ.
45
Table 3-3: The composition of phosphate buffer solutions.
85 % H3PO4, ml
KH2PO4, g
Na^HPO^, g
Na3P04, g
Measured pH
0.99
6.794
2.5
0.61
6.7983
2.75
0.34
6.8023
3.05
0.12
6.8095
3.43
0.05
6.8103
6.8133
3.7
4.43
6.7312
0.0453
4.73
6.463
0.107
5
6.0688
0.25
5.42
5.232
0.5488
5.84
4.6136
1.0166
6.17
2.7122
1.415
6.56
1.1775
1.9653
7.06
0.5317
2.1866
7.48
0.1553
2.3198
8.04
0.0256
2.3677
2.363
8.77
9.21
! -.
2.3614
0.0771
9.59
2.2292
1.868
10.2
2.0447
0.4343
3.1617
10.64
11.79
*The final volume of all buffer solution is 500 ml, and ionic strength is 0.1.
Black rubber stopper
46
Curved matel tube,
connected to syringe
CeU holder
- Cuvette
PMT
Light proof box
Figure 3-1: Schematic diagram of setup for chemi luminescent measurement in static
mode.
CHAPTER 4
THE CHEMICAL PROPERTIES OF
POTASSIUM SUPROXIDE
Comparison Studies
Since it is a well-documented oxidant for the chemiluminescent reactions of
luminol, H2O2 was used as a reference to evaluate K02's capability as an oxidizer
producing chemiluminescence.
At the same concentration (1 M), 40 \x\ each of aqueous H2O2 solution (a) and
K02(b) were reacted with 2.5 ml of lO'^M luminol. The chemiluminescences were
recorded by a flatbed recorder. The signal profiles are shown in Figure 4-1. At least three
injections were used to obtain the average peak heights for (a) and (b). The intensities in
term of peak heights can be calculated as
I. = 53.5 / 100 X 0.003 X 100 mv = 0.1605 mv
lb = 70.5 / 100 X 0.1 X 500 mv = 35.25 mv
lb /I, = 35.25 7 0.1605=219.6
Here, 53.5 is the peak height in centimeters, 100 is the scale height in centimeters which
corresponds to the signal scale of 100 mv, and 0.003 is the sensitivity of the
photomultiplier tube. Figure 4-1 clearly demonstrates that the chemiluminescence from
47
48
the reaction of luminol and KO2 emitted faster, disappeared faster with greater increased
intensity over the reaction with HjOj. Using the same experimental parameters, further
examinations shown that 5.0 x 10"' M luminol was detectable with H2O2, but 5.0 x 10"
M with KO2. The quicker chemi luminescence from the superoxide system seems more
desirable for quantition of reactions emitting chemiluminescences, and KO2 probably has
the greater potential to make P-lactam antibiotics chemiluminescent compounds.
These results indicate that the reaction mechanism with KOj is different from that
with H2O2. To increase the chemiluminescence by more than two magnitude, a significant
lowering of activity energy is necessary. It seems that the superoxide anion decomposes
into other reactive species.
Solvent Selection for Potassium Superoxide
KO2 solutions with a concentration of 0.1 M were made by dissolving 177.5 mg
of KO2 in 25 ml of various solvents just before the chemiluminescent experiments. These
solutions were evaluated using 10"^ M luminol injections made every 15 minutes. The
experimental parameters were the same as in the comparison experiments, and the
stability of chemiluminescent emissions was evaluated.
Clearly, water is not a suitable solvent for KO2 because of its extremely high
reactivity towards KO2. In CH3CN, KOjWas able to oxidize luminol and emit
chemiluminescences with similar intensities and peak profiles to KO2 in H2O. However,
the signal reproducibility was not good. Although CH3CN is miscible with HjO, the
49
mixing status is more variable following injection than is the mixing of the two solutions
in water.
CH3OH was also investigated as a solvent for KO2. The intensity of
chemiluminescence grew abruptly, but disappeared gradually. This is probably due to the
relative low rate of releasing KOj from CH3OH into the water. Even though the short-
term signals were reproducible, the chemiluminescent emissions of repeated injections
decreased significantly over time, as shown in Figure 4-2.
As nonpolar solvents, n-butanol and methylene chloride can barely dissolve KO2,
and no chemiluminescent signal was observed. Furthermore, these solvents are not
completely miscible with water.
Unfortunately, all of these solvents can not preserve the activity of KO2 over two
hours. Even when KO2 was dissolved in the mixed solvents like CH3CN-CH3OH,
CH3CN-H2O, and CH3OH-H2O, a similar loss of activity of KOj was observed. The
typical correlation between the time and chemiluminescent intensity is shown in Figure
4-3.
To increase the solubility of KO2 in CH3CN, 1 8-crown-6-ether was added to give
a saturated solution. After standing over night and centrifuged as described in chapter 3,
the saturated KO2 reagent was tested by reacting with luminol. The results were very
positive, the chemiluminescent intensity was greatly increased due to the high
concentration of KOj, and the signal was stable for at least 24 hours. More encouragingly,
fresh KO2 and crown ether can be added periodically to keep the KO2 amount constant.
50
and the reactivity of this reagent was sustained. The concentration of KO2 was found to
be 0.4386 M as determined by atomic emission spectroscopy (Zeeman 5100, Perkin-
Elmer, Norwalk, CT, courtesy to Dr. Bergeron).
51
n.
OTJ
xsry
CTT
SL
oP
fczQi
Figure 4-1 : The chemiluminescence of luminol following the oxidation by (a) H2O2 and
(b) KO2. Experimental parameters: High Voltage (HV) of Photomultiplier Tube (PMT) is
910 volts, Time Constant is 0.25, Suppression Background is set at high, recorder speed
is 1 cm/min. Sensitivity of PMT and Signal Scale of recorder is adjusted according to
varied intensities. In (a) Sensitivity = 0.003, Signal Scale = 100 mv. In (b) Sensitivity =
0.1, Signal Scale = 500 mv.
52
>. ' ■'..
Figure 4-2: The chemiluminescence of luminol reacting with KOj in CH3OH. (a) at
starting time, (b) two hours later, (c) three hours later.
53
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CHAPTERS
CHEMILUMINESCENCES AND p-LACTAM ANTIBIOTIC
Introduction
In the last few years, quantitative studies of P-lactam antibiotics by
chemiluminescences were based on either their enhancement [100, 101] or inhibition
[102] of the luminol system. In this chapter the chemiluminescent emission following the
interaction of KOj with 13 p-lactam antibiotics was investigated as a potential analytical
tool.
The chemiluminescent intensities of antibiotics were determined at optimized
parameters (pH 4.5-7.0. 950 volts across the PMT). 50 ml of saturated KO^ solution was
injected into 2 ml of 1.0 mg/ml antibiotics in 100 ml deionized water. All aqueous
antibiotic solutions were prepared just before the detection to minimize the hydrolysis
effects. The materials and methods were as described in the chapter 3,
Six of the antibiotics studied gave no measurable signal, while the other seven
emitted differently intensified chemiluminescence. The chemiluminescent intensities of
the antibiotics are compared in Figure 5-1. The strongest chemiluminescence was
observed with penicillin G. The linear dynamic range of 0.01-0.1 mg/ml is shown in
Figure 5-2. Dicloxacillin in the concentration range 0.2 to 1.0 mg/ml also gave a linear
54
55
relationship, as shown in Figure 5-3. 30 % H^Oj had been used to oxidize all the
antibiotics but no signal was observed.
Probe of Maximum Emission Wavelength
The maximum emission wavelength of the chemiluminescence following the
oxidation of 10 mg/ml penicillin G by the saturated KO2 reagent was measured at 535 nm
by using a SPEX Photocounting Fluorescence Detector Courtesy of Dr. Winefordner.
After turning off the excitation source, the fluorescence detector was ready for
chemiluminescence determination. The injection of KO2 was made at emission
wavelengths of 400, 450, 520, 530, 540, 600, 700, and 800 nm. The intensity observed at
535 nm was the highest. The chemiluminescenct spectrum between 530-590 nm is shown
in Figure 5-4. To validate the detecting system, the spectrum of 10"* M luminol (Figure 5-
5) was observed by reacting it with 30% H^Oi, this data is consistent with the literature
[77]. The experiments here demonstrate that the maximum emission from the reaction
between penicillin G and KO2 is at 535 nm.
Discussion
There are some unique characteristics produced by the reactions of p-lactam
antibiotics with KO2. First, in these experiments seven of the thirteen p-lactam antibiotics
generated chemiluminescences at pH 4.5-7.0 rather in the more usual alkaline media. It
should be remembered that these antibiotics will hydrolyze at extremely acidic or basic
pH values [103]. Second, the maximum emission wavelength of chemiluminescence
56
following the oxidation of penicillin G by KO2 was observed at 535 nm, which is
different from the 425 nm of 3-aminophthalate (from luminol) [77], 275-425 nm range of
S2 [89], 260-480 nm of SO^ [91]; but is closer to the 420-500 nm range of N-
methylacridone (from lucigene) [84] or 562 nm of oxyluciferin (from luciferin) [87].
Third, the chemiluminescences of the emitting antibiotics disappeared very quickly, the
typical full width at half maximum (FWHM) was 2 sec (Figure 5-6); the slowest
emission, from ampicillin, had a FWHM of 7 sec (Figure 5-7) and probably a diode-array
detector is needed to observe the frill spectrum.
However, there are still several questions remaining to be answered.
1. When penicillin G was dissolved in an aprotic solvent like CH3CN, even after
oxidation by the KO2 saturated solution, no chemiluminescent signal was observed. In
dry CH3CN, KO2 exists in the form of superoxide anion radical. This may indicate that
without dismutation reactions with water or other protic solvents, superoxide can not be
transferred into the reactive species, and so the question arises what is the reactive species
generated from the superoxide dismutation reactions? Since H2O2 is not able to generate
chemiluminescence after oxidizing any of the p-lactam antibiotics, a possible candidate
of this reactive specie is 'Oj. Further experiments are conducteded to prove its existence.
2. Why do the six antibiotics including azetidinone, sulbactam, clavulanic acid,
methicillin, penicillin V, and cefotaxime not produce chemiluminescence in the reaction?
Why do the other seven antibiotics gave chemiluminescences of different intensities?
What are the chemiluminescent intermediates and through which pathways do they go?
57
3. Is it possible that in this reaction the hydroxyl radical has been generated, i.e. is
the oxygen toxicity theory based on the Haber- Weiss reaction the right assumption?
To answer these questions, the degradation products of P-lactam antibiotics in the
reactions with KOj needed to be identified, and a DO2 experiment needed be designed to
prove the existence of 'O2 in the superoxide dismutation reaction.
■■■'.''-'- '■ ■ ,V if. \ " 1^
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CHAPTER 6
MECHANISTIC STUDIES
Penicillin G was chosen as an example to investigate the mechanism of the
chemiluminescence of P-lactam antibiotics interacting with potassium superoxide.
Various experiments were conducted on the aqueous penicilhn G sample and the sample
containing the products generated by oxidizing penicillin G with potassium superoxide.
These experiments include nuclear magnetic resonance (NMR) assay, thin-layer
chromatography (TLC), and high performance liquid chromatography (HPLC) using a
ultraviolet (UV) spectrophotometer, a photodiode array (PDA) detector, and a mass
spectrometry (MS) detector. In all these experiments, solid KOjWas utilized instead of
the saturated KOj reagent (K02-acetonitrile-18-crown-6-ether) in order to simplify the
experiments. A DO2 experiment was also run to prove the generation of singlet oxygen in
the suproxide dismutation reaction. ,
',. ' '^ »' V ! j" The Deuteration Experiment
Stauff et al. [104, 105] proposed that the spontaneous disproportion of superoxide
generating 'O^, rather than ground state oxygen CO^), as shown in equation (1). This
proposal had received support from the calculations of Knoppenol [106].
65
66
Khan [94, 95] had also proposed that superoxide is a precursor of 'O2, although he
believes this occurred by a direct electron transfer reaction [equation (2)].
Oj- ^ e- + 'O2 (2)
Nilsson and Keams [98], however, based on experiments injecting DjO rather than H2O
into the dry dimethyl sulfoxide (DMSO) containing KOj, rejected the suggestion that
superoxide serves as a direct precursor of 'Oj. The major question remaining in their
investigation is the fact that even though it can be generated from KO2 after injection of a
small amount of D2O or H2O, 'O2 will be mostly surrounded by DMSO, not by D2O or
H2O because both react with superoxide through the dismutation reaction. Comparing the
30 usee lifetime of 'Oj in DMSO with its 20 usee in DjO and its 2 ^sec in H2O it is not
surprising that no difference was observed. Also, whether the frace amounts of singlet
oxygen produced in this system is enough to oxidize the 'O2 acceptor tetramethylene
(TME) is uncertain, and thus it is possible that no oxidation signal of TME could be
detected in the KOj system adopted by Khan's experiments.
Since a relatively strong chemiluminescence had been obtained in the reaction of
aqueous penicillin G interacting with KO2, D2O was used instead of H2O to dissolve
penicillin G, and to examine the deuterium effect on the chemiluminescent intensities. In
view of the fact that in DjO 'O2 has more time to react with penicillin G, an enhancement
of chemiluminescence supports the concept that the 'Oj is generated in the superoxide
dismutation reaction. 4.0 mg/ml of penicillin G solutions were prepared in pure DjO,
70% D2O-30% H2O, 50% D2O- 50% H2O, and pure H2O. The chemiluminescences of 2
67
ml of these solutions were tested by injection of 50 (jl of KOj, which is contained in a
saturated solution made of 18-crwon-6-ether and acetonitrile. The experimental
parameters were set as the following: Gain (PMT/HV) is 8.8, Sensitivity is set at auto,
Time Constant is 5, and Suppression Background is set at high. The differently
intensified chemiluminescences are described in Table 6-1, and the chemiluminescent
profiles of penicillin G in the mixture solvents with varied DjO to HjO ratios (fi-om pure
DjO-the top to pure HjO-the bottom) are demonstrated in Figure 6-1. Inspection of Table
6-1 clearly shows an approximately 9-fold enhancement of chemiluminescent emission of
penicillin G by replacing Rfi with DjO as the solvent. Also lowering the ratio of D^O to
HjO was correlated with decreased intensities of chemiluminescences. Another
observation really attracts attention. When the high voltage of photomultiplier tube
(PMT) was adjusted to 980 volts, the time constant was set at 0.25 to catch the weak and
short-life emissions. After the first injection of KO2 into the solution of penicillin G in
pure DjO, the huge numbers of photoelectrons collected by the PMT outscaled the
detector. Characteristically, even after a second, or third injection of KOj into the same
reacting cell, a significant signal was still observed, although their intensities dramatically
decreased. This is totally different from the case of the penicillin G in pure HjO, where a
much weaker chemiluminescence from the first injection of KOj was detectable, but no
signal was observed after the second injection of KOj. This phenomenon fiirther suggests
that 'O2 can be generated in the superoxide dismutation reaction. In this detection system,
the syringe needle was connected to a long, curved metal tube, which is immersed in the
68
center of the cell to prevent the light leakage caused by injection. After injection, KO2
diffuses radially from the center to the cell wall. Most penicillin G reacts with the KO2
and the concentration of penicillin G is decreased, especially in the center. KO2 from the
second or third injection has to fravel relatively longer distance to react with the
remaining penicillin G. The singlet oxygens produced from the second or third injection
of KO2 into the D2O therefore have more opportunity to react than those in H2O because
of the longer lifetime of 'O2 in D2O. In the contrast, 'O2 formed in H2O can not fravel far
enough to react with penicillin G away from the center. The repeated injections is an
advantage of investigations in the static mode.
The results presented in this experiment sfrongly reinforce that in the contrast of
the Haber- Weiss reaction, the 'Oj can be generated by superoxide dismutation reaction,
which can occur either spontaneously or when catalyzed by SOD with great rate. It
indicates that superoxide maybe must not have time to fiuther react with hydrogen
peroxide to produce hydroxyl radicals and cause oxygen toxicity, as described by the
Haber- Weiss reaction. Even though it is catalyzed by fransition metals, which are
available in biological systems, the Haber- Weiss reaction will face competition from the
Fenton type reaction [equation (3), (4)].
O2- + Fei° ^ Fe" + O2 (3)
Fe° + H2O2 ^ Fe°' + OH + OH' (4)
69
Equation (4) is consistent with the fact that plenty of H2O2 has been generated ahnost
instantly by the superoxide dismutation reaction. It is already claimed that the evidence
for the formation of OH is overwhehning [107, 108,109, 110]. This does not, of course,
preclude the formation of reactive oxygen species in additional to OH, for example 'O2 in
equation (3). Taking account of the fact that hydroxyl radicals may also come from the
Fenton type reaction as well as the Haber- Weiss reaction, and the 'O2 is capable of
damaging the same biological components as is superoxide, not all the oxygen toxicity
should be rationalized by the Haber-Weiss reaction. The toxic species should be specified
in order to better understand the pathology, which causes the diseases, and to develop
new therapeutic strategies as well as new antioxidants. In the experiments discussed
above, it is strongly suggested that 'O2 well deserves more attention, and it is probably
the prime oxidant in various diseases caused by oxygen toxicity.
NMR Consideration
Two sample of 1 5 mg of penicilhn G ( or penicillin V) in 0.75 ml deuterium oxide
were made, one of them was reacted with 10 mg KO2. The 300 MHz 'H-NMR, 75 MHz
"C-NMR and Attached Proton Test (APT) were obtained by a GEMINI-300 NMR
spectrometer at room temperature. 3-(Trimethylsilyl) propionic-2, 2, 3, 3-d4 acid, sodium
salt (TSP, Aldrich) was used as internal NMR standard and the HDO peak appeared
around 4.8 ppm from TSP. These NMR assays are used to detect the structural alternation
70
before and after the oxidation of penicillin G or penicillin V by KOj and to check the
purity of these two drugs.
NMR Spectra of PenicilHn G
Figure 6-2 is the proton NMR spectrum of potassium penicillin G in Dfi. The
interpretation of the spectrum is given below.
^^^f Chemical Shift (ppm) Position
^^9\/"^— « O 1.501 (s), 1.570 (s) 1,2
^^3C s^^-^\ // 3.615, 3.630 (ABq,J= 14 Hz) 10
HN--^ 4.247 (s) 4
1° 5.430 (d, J=3.9 Hz) 6,7
peniciUinG f^\ 5.493 (d, J-3.9 Hz) 6,7
4, f 7.278-7.392 (m) 12,13,14
Figure 6-3 depicts the '^C-NMR spectrum of potassium penicillin G in DjO. The
assignments are tabulated below.
. ;;• .i ......?..■ •^'-. H
' .* Chemical Shift (ppm) '•' '-J Positbn
26.594,30.843 ' 1,2
42.088 lb
58.190 7
64.518 3
66.779 6
73.305 4
127.619 14
129.137,129.486 12,13
134.600 ll'
174.345, 174.497, 174.786 5, 8, 9
71
The results here of 'H-NMR and "C-NMR spectra are similar to these presented
in a study of penicillins and cephalosporins [111].
Compared with Distortionless Enhancement by Polarization Transfer (DEPT),
APT is less sensitive and does not distinguish between CH3 and CH peaks (both down) or
between CHj and quaternary C peaks (both up). However, in the case of penicillin G and
penicillin V, the partial information obtained by APT is sufficient for interpretations. As
shown in Figure 6-4, the CH3 peaks at position 1, 2 and CH peaks at position 4, 6, 7, 12,
13, 14 are down. Since the stronger peaks at 127.619 ppm, 129.137 ppm, and 129.486
ppm obviously belong to the aromatic carbons, the other down peaks are easily identified.
For the up peaks, the peaks at 174.786 ppm, 174.497 ppm, and 174.345 ppm are clearly
from carbonyl groups; the peaks at 134.600 ppm and 64.5 18 ppm refer to the quaternary
carbon of aromatic ring and alkanes respectively. Subsequently the peak at 42.008 ppm is
identified as CH2 at position 10.
The degradation products of penicillin G generated by reaction with potassium
superoxide were also examined by NMR spectroscopy without isolation as shown in
Figure 6-5 ('H-NMR spectrum), and Figure 6-6 ('^C-NMR spectrum). The APT spectra
of degradation products were not readable and so are not presented. Interestingly, the
NMR signal at position 10 in Figure 6-6 seems to have disappeared. Probably penicillin
G lost the CH2 at position 10 after oxidation by KO^. However, there is a peak at 42.589
ppm close to 42.088 ppm (position 10 in Figure 6-3) in the '^C-NMR spectrum, which
may suggest that the electronic environment has changed but CH2 still exists. More
72
experiments are needed to clarify the status of the methylene, which is between the
carbonyl and benzyl moiety on penicillin G.
Meanwhile the comparison between the NMR spectra of the phenyl ring before
and after the oxidation by KO2 draws attention. If the hydroxyl radical is generated by the
interaction between superoxide and hydrogen peroxide, which is produced by the
superoxide dismutation reaction, the hydroxylate aromatic compounds should be
identified by NMR spectra since the electrophilic hydroxyl radical adds readily to the
aromatic nucleus (K = 3 ~ 8 x lO'M" S') [112]. No such hydroxylate compounds are
demonstrated in Figure 6-5 and Figure 6-6. There is no disubstitution or chemical shift of
the aromatic ring to suggest the hydroxylation. Hence, no conclusion can be drawn that
the hydroxyl radical is formed by the Haber- Weiss reaction in this superoxide system.
NMR Spectra of Penicillin V
As shown in Figure 5-1, penicillin G emitted the strongest chemiluminescence
after the oxidation by KO2 and surprisingly no chemiluminescent signal was observed
with penicillin V under the same reacting conditions. Considering the fact that the only
structural difference between penicillin G and penicillin V is the "phenoxyl methyl"
moiety of penicillin V, the oxidation of potassium penicillin V in D2O was examined by
NMR spectroscopy by comparing the spectra following the oxidation of penicillin G and
penicillin V by KOj.
The proton NMR spectrum of penicillin V in D^O is shown in Figure 6-7, and the
interpretation is described as below.
73
/
/
X
0==\
OH
./T
10'
— O
\
\
11=12
14
penicillin V
Chemical Shift (ppm) Position
1.500,1.513(8)
u
4.260 (s)
4
4.469 (ABq, J=16.2 Hz)
10
5.520 (d, J=4.2 Hz)
6,7
5.569 (d, J=3.9 Hz)
6,7
6.837-7.273 (m)
12,13,14
Figure 6-8 is the carbon NMR of penicillin V in DjO. The peaks are assigned as
the followings, which are in agreement with the literature values [113].
Chemical Shift (ppm)
26.548,31.238
57.492
64.670
66.49 P
66.613"
73.260
114.841
122.384
130.002
156.908
170.643
174.285"
Position
1,2
7
10
3
4
12
13
14
11
5
A9
"the peaks with chemical shifts of 66.491 and
66.613 ppm are overlapping and they are
differ enciated by APT (Figure 6-9).
"the peaks of carbons at position 8 and 9 are
overlapping at 174.285 ppm.
74
In case of penicillin V, APT is very useful to identify carbon 3 at 66.492 ppm (up)
and carbon 6 at 66.597 ppm (down) as shown in Figure 6-9. The degradation products of
penicillin V after the oxidation by KO2 were also studied by proton NMR (Figure 6-10)
and carbon NMR (Figure 6-1 1). Both showed no structure alternation on the aromatic
ring, and contrary to penicillin G the CHj at position 10 remains intact.
Conclusion
As discussed in chapter 1 (equation 7), the quantum yield of chemiluminescence
is associated with Or, the yield of product; O^s.the number of molecules entering the
exited states; and Op the fluorescence quantum yield. Therefore, the generation of
chemiluminescent products is the first step to emit chemiluminescence. It seems that
unlike penicillin G, penicillin V did not carry out this first step and no chemiluminescent
signal was observed. The NMR data here suggests that the CH2 at position 10 be
correlated with the chemiluminescent emissions, and no hydroxylate aromatic compound
was found to support the possibility that hydroxyl radical was generated by the Haber-
Weiss reaction in the current superoxide system.
TLC Analysis
Introduction
The TLC procedures have been used to allow the simple and rapid separation and
identification of the different (spontaneous, chemical and enzymatic) degradation
products of penicillins and cephalosporins [114]. After testing several spray reagents, Lin
75
and Kondo [115] found that the iodine-azide reagent was the best for detection of
penicillin G, producing a yellow color on the TLC plate; the vanillin-phosphoric acid
mixture was the best for the detection of streptomycin (brown) and dihydrostreptomycin
(brown); ninhydrin was best for kanamycin (purple) and fradiomycin (purple). Since the
NMR spectra had indicated that numerous degradation products of penicillin G were
generated after the oxidation by KOj (Figure 6-5 and 6-6), these three reagents (iodine-
azide, vanillin-phosphoric acid, and ninhydrin) were utilized in the TLC analysis to
separate the hydrolysis products from the oxidation products or other derivatives of
penicillin G. It is anticipated that sufficient samples would be obtained to conduct
experiments using mass spectrometry after separation and purification.
Experimental
TLC plates (1 inch x 3 inches) of silica gel were used for separation. Five
different solvent systems were tested: (A) n-butanol-water-ethanol-acetic acid
(5:2:1.5:1.5); (B) n-butanol-water-acetic acid (4:1:1); (C) acetone-acetic acid (19:1); (D)
acetone-water (17:1); (E) methanol-water (50:50). Among them, solvent system (B) was
the best for separating the degradation products of penicillin G. Each TLC plate was
sprayed consecutively with the following spray reagents: (a) ninhydrin, (b) iodine-azide,
(c) vanillin-phosphoric acid. The iodine-azide reagent was prepared by dissolving 0.5076
g iodine (Sigma) and 58.9 mg sodium azide (Fisher) into 200 ml chloroform to make a
solution of 0.01 N iodine-0.02 % azide. Vanillin (Sigma) 0.9992 g was dissolved in 100
ml phosphoric acid and this solution was diluted 1:1 with methanol to form the vanillin-
76
phosphoric acid reagent. Ninhydrin 0.2010 g was dissolved in 5 ml of 10 % acetic acid,
and added to 95 ml of n-butanol to produce the ninhydrin reagent. Since spraying
vanillin-phosphoric acid mixture did not mark any new spot, as shown in Table 6-2, this
reagent was not used in the separation procedures.
Results and Discussions
Each of the aqueous penicillin G sample (sample PG) 10 mg/ml and the sample
containing degradation products of penicillin G (sample PGKOj) was made as in the
NMR experiments. Both of them were adjusted to pH 12.00 and were spotted on the same
TLC plate. Each spot was dried with pressured air and the spotting was repeated four
times to increase the amount of sample in each spot. After the first elution in the solvent
system (B), the TLC plate was dried by a hot plate, then ninhydrin and iodine-azide were
sprayed consecutively on the TLC plate. Three spots were found originating from sample
PGKO2, two spots from sample PG. The colors and the moving distances of these spots
were shown in Figure 6-12. Obviously the second spot from sample PGKO^ and the first
spot from sample PG have the same Revalue (distance solute moves/distance solvent
front moves). After the third elution, three spots of sample PGKO^ were found to have the
same Revalues as those of sample PG, as shown in Figure 6-12. This indicates that while
it was oxidized by KO^, the aqueous penicillin G was hydrolyzed as well because of the
sfrong basicity of superoxide in water [116]. Therefore, the first three spots of sample
PGKO2 after the third elution were believed to be produced from the reactions other than
hydrolysis.
77
To prepare for the mass spectroscopy experiments, 100 mg/ml of sample PGKOj
was made and put on one of the 36 TLC plates in a line spots. Each spot was dried by
pressured air and the spotting was repeated four times. The plate was sprayed after the
third elution, 4/5 of the plate was covered by a piece of paper and the other 1/5 was
sprayed by ninhydrin and iodine-azide, as shown m Figure 6-13. The invisible three lines
on the 4/5 part of the TLC plate was located by the sprayed 1/5 part of the plate, then
each line on the 4/5 part was cut off separately. After the three desired block on all the 36
plates were cut off and separated into three samples, each of them was washed by
methanol and the silica gel was filtered. The subsequent solution was concentrated into
three approximate 1.5 ml solution (solution 1, 2, and 3). Each solution was repeatedly
spotted 12 times on a TLC plate (Figure 6-13) and developed in the solvent system (B).
After the first elution and the two reagents were sprayed, two spots were found from
solution 1, one from solution 2, and none from solution 3. Possibly solution 3 results
from the running solvents or from the solvent of sample PGKO2, as its spots were
overlapping with the solvent front. These four spots were named 1 1, 12, 13, and 3.
Following the same procedures of mass production as discussed before, four solid sample
were finally obtained after filtering, and drying and were analyzed by Fast Atom
Bombardment (FAB)-mass specfroscopy. The average lengths of the time consumed
during each run are listed in Table 6-3.
One peak of m/z 105 with quite low intensity was found in one of the four
samples, as shown in Figure 6-14. This is most likely ph-C=0, however, in the other
78
three sample spectra it appears that they was enough sodium salts in the samples to
suppress other signals. The sodium salts may come from the TLC plates or from other
reagents. Although it is consistent with the results of NMR assay, in which the CHj seems
lost after the aqueous penicillin G was oxidized by K02, the peak m/z 105 in mass
spectrum of spot 13 seems unreliable and better separation methods are needed.
HPLC Detection
t,
HPLC is widely utilized to analyze the degradation products of penicillin G in
aqueous solutions because of its specificity, ease of use and high sample capacity.
However, no product generated by the oxidation of penicillin G had been studied by
HPLC. In this section the products of aqueous penicillin G following the oxidation by
potassium superoxide is investigated by HPLC using a CI 8 column.
Experimental
The HPLC experiments are conducted with the following equipment:
1) Beckman Model 11 OA pump
Beckman Model 153 UV detector
Beckman Instrument Inc., FuUerton, CA.
2) Hewlett-Packard 3392A integrator
Hewlett-Packard Co., Palo Alto, CA.
3) Waters 501 HPLC pump
Waters™ 1 996 Photodiode Array Detector
79
Waters™ 7 1 7 plus Autosampler
Millipore Corporation, Milford, MA.
4) Exsif ODS column, 150 by 4.6 mm dimension, 7 |im particle size, 100 A pore size.
Keystone Scientific, Inc., Bellefonte, PA.
5) Helium compressed gas, Gainesville Welding Supply, Gainesville, FL.
Various reference compounds were injected and the retention times were
measured in order to identify the unknown peaks, which were produced by the
degradation of penicillin G following the oxidation by potassimn superoxide. Hydrogen
peroxide was obtained from Fisher. 3,4-dihydroxybenzoic acid, 2, 4-dihydroxybenzoic
acid, 2, 3-dihydroxybenzoic acid, phenylacetic acid, p-hydroxyphenyl acetic acid, and
benzaldehyde were bought from Aldrich. Benzoic acid was obtained from Sigma.
P-hydroxybenzoic acid was obtained from Eastman (Eastman Organic Chemicals,
Rochester, NY). Salicylic acid was obtained from Mallinckroot (Mallinckroot Chemical
Works, St. Lois, New York, and Montreal). All the chemicals, including the reference
compounds and the samples, were completely dissolved using an ulfrasonic cleaner. All
the reagents utilized by the HPLC system were filtered and degassed for about 10
minutes. Prior to the experiments, the HPLC setup was washed by water, 50:50
watenmethanol, and methanol consecutively, each for half-hour. The UV detector was set
at 254 nm, 0.08 absorbance unit fiiU scale. The flow rate was 1.0 ml/min and the pump
pressure was kept below 4000 psi. The 2 mg/ml of sample PG and PGKOj were prepared
as in chapter 3.
80
Results and Discussion
30% MeOH-70% phosphate buffer (pH4.15) is good for the separation of the
hydrolysis products of penicillin G in aqueous solutions, but is not good for oxidized
sample, which is consistent with the observation of Vadino et al. [117]. Acetonitrile 0.1%
in pH 5.66 phosphate buffer was found useful for the detection of the oxidation products
of penicillin G as shown in Figure 6-15. Unfortunately, none of the retention times of
unknown peaks of sample PGKOj matched up with that of the reference compounds as
shown in table 6-4. Since the same CI 8 column was not available, the experiments using
photodiode array detector (PDA) were conducted on a different Exil'^ODS column.
Similar results were obtained as shown in Table 6-4.
Conclusion
1 . When the sample PGKOj stayed overnight huge peaks appeared hours after the
injections. Were polymers generated?
2. No benzoic acid or its derivatives were identified by this kind of detection and maybe
the amount generated is so small that it is out of the detecting limit of the UV
detector. '"'■. ' '"'■' '
3. UV detector is not suitable for the detection of the mixture caused by oxidation and
hydrolysis. Mass spectrometer is the obvious choice.
4. The HPLC buffer of sodium salts must be replaced by ammonium buffers or other
nonmetal-ion solutions since in the TLC analysis vast amounts of sodium salts
suppress the signal for mass spectroscopy.
81
HPLC/ES I-MS and HPT C/APCI-MS Analysis of the nemdation of Penir.illin O
Following the O xidation hv Potassium Sup ernyidt^
Introduction
Electrospray ionization mass spectrometry (ESI-MS) was first introduced by
Yamashita and Fenn [118, 119] in 1984. At approximately the same time, a veiy similar,
independent development was reported by Aleksandrov and coworkers [120]. Actually,
electrospray as a source of gas-phased ions and their analysis by mass spectrometry was
proposed much early by Dole [121], however Dole's experiments were too narrowly
focused on the detection of polymeric species, which are not themselves ionized in
solution, and the experimental results were not convincing [122].
Since the end of 1980s, ESI-MS has developed at a tremendous pace and
established itself as an outstanding method for biochemical applications. It has permitted
new possibilities for mass spectrometric analysis of high-molecular-weight compounds of
all types, including proteins, nucleotides, and synthetic polymers. Other analytical
techniques do not provide the same level of detailed information regarding molecular
weights and structures from extremely small amounts of material. Compared to the other
mass spectrometric ionization techniques, ESI has three superior features. First, ESI has
the truly unique ability to produce extensively multiply charged ions, which may be
analyzed on virtually all types of mass spectrometers. Second, the samples analyzed by
ESI-MS must be introduced as a liquid phase. This results in a natural compatibility of
82
ESI with many types of separation techniques. Third, the extreme "softness" of ESI
process allows the preservation in the gas phase of noncovalent interaction between
molecules as well as the study of three-dimensional conformation. For earlier ionization
methods such as fast atom bombardment (FAB) and plasma desorption, abundant energy
is applied in a highly localized fashion over a short time. These methods lead not only to
ion desolvation but also to fragmentation or even net ionization, that is , the creation of
ions from neufrals. As for ESI-MS, in which the desolvation is achieved gradually by
thermal energy at a relatively low temperature as it is a far softer technique.
There are three major steps in the production of gas-phase ions from electrolyte
ions in solution by elecfrospray: (1) production of the charged droplets at the ES capillary
tip; (2) shrinkage of the charged droplets by solvent evaporation and repeated droplet
disintegrations, leading ultimately to very small highly charged droplets capable of
producing gas-phase ions; and (3) the actual mechanism by which gas-phase ions are
produced from the very small and highly charged droplets. Kebarle and Tang [123]
described the first two steps in Figure 6-16. As shown in this schematic representation of
the charged-droplet formation, a high voltage of 2-3 KV is supplied to the metal capillary
which are typically 0.2 mm o.d. and 0.1 mm i.d. and located 1-3 cm from the counter-
electrode. Penetration of an imposed electric field into the liquid leads to formafion of an
electric double layer in liquid. Enrichment of the surface of the liquid by positive
elecfrolyte ions leads to destabilization of the meniscus and formation of cone and jet
83
emitting droplets with excess of positive ions. Charged droplets shrink by evaporation
and spht into smaller droplets and finally gas-phase ions.
More recently, HPLC/ESI-MS has gained widespread recognition as a powerful
analytical tools for drug metabolism and pharmacokinetic studies because it permits the
separation and ionization of polar nonvolatile or thermally labile compounds, such as
drugs, conjugated metabolites, peptides or DNA adducts. In addition, these techniques
facilitate qualitative and quantitative studies on the parent compounds and their polar
metabolites. Given its attractive advantages such as good sensitivity, reliability, and
specificity for a wide variety of compounds with minimal sample handling, HPLC-MS
has replaced GC-MS, which requires sample derivatization for polar metabolites.
A series of ionization sources compatible with HPLC is available, which include
thermospray ionization (TSP), electron impact (EI) or chemical ionization (CI) with
particle beam interface (PB), continuous-flow fast atom ionization (CFFAB) and
atmospheric pressure ionization (API) using a heated nebulizer interface (APCI). Most of
those methods have their limitations. TSP and APCI require critical control of the
vaporizer temperature during analysis, and thermal degradation of labile molecules may
occur. CFFAB can only accept low flow rates, and requires the presence of a matrix to
assist ionization, thereby affecting the ion-current stability and making the method
susceptible to interference from background ions. However, HPLC/ESI-MS is now fi-ee
of most major technical problems and has become the method of choice for drug
metabolism and pharmacokinetic research, as reflected in the impressive number of
84
applications published in the last few years. More encouragingly, a triple-quadrupole
mass spectrometer is capable of performing MS/MS such as neutral-loss scan, parent-ion
scan or product-ion scan. Therefore, besides the molecular weight, HPLC/ESI-MS/MS
(or MS") can give complex mixture analysis, structural elucidation, combinatorial
screening, fast chromatography, picogram quantitation and protein sequencing. For
example, in addition to MS/MS capabilities, the LCQ LC/ MS" Detector (octapole-
octapole mass spectrometer, Finnigan MAT, San Jose, CA) also provides standard
MS/MS/MS. . .. MS" (n=l-10) for structural elucidation capability. The selectivity of MS"
means that a compound can be fragmented, and the resulting fragments fiirther isolated
and analyzed to yield structural information about complex molecules. The results
presented here confirm that HPLC/ESI-MS/MS is an outstanding tool for pharmaceutical
analysis.
Experimental
AH data were obtained on a Finnigan MAT (San Jose, CA) Benchtop LCQ
LC/MS" Detector (external ionization ion trap) equipped with both an electrospray
ionization (ESI) ion source and an atmospheric pressure chemical ionization (APCI) ion
source. These sources were interfaced to a Hewlett-Packard (Palo Alto, CA) 1090 (Series
II) high pressure liquid chromatograph (HPLC). The effluent from the HPLC column was
typically split prior to the ion source.
Chromatographic separation was achieved on a Keystone Scientific (Bellefonte,
PA) Exil"^ ODS (C-18; 150x4.6 mm dimension; 7 ^m particle size; 100 A pore size)
85
HPLC column utilizing a reverse phase gradient. Two mobile phases were used. Mobile
phase A was 0.1% acetic acid in 50:50 water: acetonitrile and mobile phase B was 0.01 M
ammonium acetic acid (pH 5.72) with 0.1% acetonitrile in water. The flow rate was 1
ml/min and the loop size is 5 jil. After 1 mg/ml and 0.1 mg/ml penicillin G were oxidized
respectively by solid KO2, the degradation of penicillin G was analyzed via direct
infusion between HPLC and ESI-MS at room temperature. A number of different
gradients were utilized as described in table 6-5.
Results and Discussion
A number of derivatives and their isomers of penicillin G were separated and
identified by HPLC/ESI-MS/MS after the interaction between aqueous penicillin G and
solid potassium superoxide. Most of these can be classified into the products of
hydrolysis, oxidation, recombination reactions including dimerization, and
chemiluminescent reaction.
Among these compounds benzylpenilloic acid (MW 308), benzylpenicillenic acid
(MW 334), benzylpenillic acid (MW 334), and benzylpenicilloic acid (MW 352) were
found which arise fi-om the base-catalyzed hydrolysis of penicillin G in aqueous
solutions. The chromatograms and mass spectra of HPLC/ESI-MS/MS of these four
compounds plus penicillin G and the interpretations of mass spectra are presented in
appendix A. The existence of these compounds was supported by the degradation
pathway of penicillin G proposed by Kessler et al [124].
86
The oxidation of penicillin G and its derivatives occurred exclusively on the
sulfur atom. Derivatives like compound 312 (a product of the oxidation of penicillamine,
which is one of the products of hydrolysis of penicillin G), bertzylpenilloic acid sulfoxide
(MW 324), benzylpenillic acid sulfoxide (MW 350), and benzylpenicilloic acid (MW
368) are examples. Two isomers of penicillin G sulfone were also found and the
experimental results and their interpretations are recorded in appendix B.
The recombination of the derivatives of penicillin G resulted in several new
compounds, such as the dimer of penicillamine (MW 296), compound 440 (a product of
recombining of compound 269 and benzylpenilloaldehyde, one of the hydrolysis products
of penicillin G), compound 632 (a product of penicillamine dimer and ben2ylpemcilloic
acid), and compound 642 (a product of benzylpenilloic acid and benzylpenicillenic acid).
The HPLC/ESI-MS results and identifications of these compounds are in appendix C.
The structures of these compounds and the mechanism of dimerization are simunarized in
table 6-6. It is believed that the thiol (penicillamine) loses a proton, and then it is oxidized
by oxygen to form a free radical and radical coupling generates the dimer [125]. Singlet
oxygen is probably involved in this process.
The identification of benzoic acid, compound 336, and compound 511 (a product
of compound 336 and benzylpenilloaldehyde) appears to support that a chemiluminescent
reaction was involved in the degradation of penicillin G following the oxidation by
potassium superoxide. The data of mass spectra of these three compounds and their
interpretation are shown in appendix D.
87
In addition, compound 269 and compound 353 were identified by their mass
spectra, as described in appendix E, but their sources are unknown.
Figure 6-17 illustrates the total chromatogram between 0-26 min of HPLC/ESI-
MS of sample PG and sample PGKOj while the HPLC gradient is 100% mobile phase B /
0% mobile phase A. The top chromatogram demonstrates the hydrolysis of penicillin G
in the aqueous solution. The peak with the retention time of 22.15 min is most likely
penicillin G and the other major peaks at 19.94 min, 20.08 min, 20.91 min, 22.59 min are
the derivatives of penicillin G after hydrolysis. All of these were identified by their ESI-
MS/MS and verified in the gradient, 100% mobile phase A / 0% mobile phase B. The
retention time in different gradients, the structures, the molecular weight, and the mass /
charge ratios of the parent ions are listed in table 6-7. The bottom chromatogram was
acquired from sample PGKOj and it clearly shows that during the process of oxidation of
penicillin G by potassium superoxide, the hydrolysis of penicillin G occurs as well. This
is indicated by the observation that the intensities of the peaks at 19.88 min (the
overlapping of peaks at 19.94 min and at 20.08 min in the chromatogram of sample PG)
and at 22.59 min increased significantly, however, the peak of penicillin G at 22.15 min
seems to have disappeared in the chromatogram of sample PGKOj. The experimental
results are consistent with the fact that superoxide is a strong base in aqueous solution
[1 16] and once it entered aqueous solutions, it becomes a catalyst for the hydrolysis of
penicillin G; thus, the degradation rate was increased. A close-look at the chromatogram
of sample PGKOj, as shown in Figure 6-18, shows that there are two major peaks with
88
retention times at 20.56 min and 20.81 min, which have no equivalents in the
chromatogram of sample PG. The interpretation of the HPLC/ESI-MS of those two peaks
suggests that they are ben2ylpenicillic acid sulfoxide and benzylpenilloic acid sulfoxide,
as shown in table 6-8. Actually, by using different HPLC gradients, numerous sulfoxide
of the derivatives of penicillin G were found, which were summarized in Figure 6-19, and
the pathways of oxidation are specified.
Consider the chemiluminescences of thirteen p-lactam antibiotics. Since there are
some sulfur compounds like Martin's sulfiirane, which becomes chemiluminescent
through sulfoxide or sulfone in the presence of DBA (9, 10-dibromoanthracene) [126], or
rubrene [127], perhaps sulfoxide is the right intermediate to give chemiluminescence
from P-lactam antibiotics. This is consistent with the fact that no chemiluminscence was
observed with the reactions with azetidinone, clavulanic acid, and sulbactam. They either
have no sulfur, or the sulfur is aheady oxidized to sulfone, as shown in table 6-9. This
concept is not supported by the fact that as shown in table 6-9, methicillin, penicillin V,
and cefotaxime did not emit chemiluminescence, even though they have sulfur in their
structures. It is also impossible to rationalize the differently intensified
chemiluminescences of penicillin G, dicloxacillin, piperacillin, ampicillin, cephalothin,
amoxicillin, and hetacillin in table 6-10 when such a proposal is adopted. Furthermore,
the maximum wavelength of chemiluminescence emitted from SO2 ranges from 260 nm
to 480 nm; for S^, it is 275-425 nm; for HSO, it is 360-380 nm (table 1-4). Obviously, all
of them don't match with the 535 nm of penicillin G, as determined in chapter 5.
89
Therefore, the claim that the chemiluminescence emitted by p-lactam antibiotics came
from the oxidation of sulfur is not supported. On the other hand, the existence of benzoic
acid, compound 336, and compound 511 in the degradation of penicillin G was proved by
their HPLC/ESI (or APCI)-MS/MS and the involvement of singlet oxygen in the
chemiluminescent reaction of the penicillin G reaction was verified by the DjO
experiments. It is proposed that the autoxidation initiated by singlet oxygen is the
mechanism of the chemiluminescences emitted by P-lactam antibiotics. As shown in
Figure 6-20, the dismutation of potassium superoxide generates singlet oxygen ('Oj) and
as a strong base in aqueous solutions, superoxide facilitates the removing the a-proton by
'Oj to form dioxetanone and 6-aminopenicillanic acid (6-APA). Dioxetanone then loses
carbon dioxide and generates the excited phenylcarbonyl cation, which emits the
chemiluminescence at 535 nm by relaxation. Phenylcarbonyl cation can either recombine
with 6-APA to produce compound 336, or be ftirther oxidized by 'O^ (or H^Oj) to
produce benzoic acid. Compound 51 1 is formed when compound 366 reacts with
benzylpenilloaldehyde. Compound 336 and compound 511 are found by electrospray
ionization (ESI) mass spectrometry. The mass spectra of individual peaks are also
obtained to further confirm the structures (MS/MS). The presence of benzoic acid has
been proved by negative atmosphere pressure chemical ionization (APCI) mass
spectrometry. This type of reaction has two characteristic features: (1) an acidic a-proton
whose removal by 'O2 leaves a special stabilized carbon radical, (2) a good leaving group
expelled by the attack by peroxide to give a dioxetanone. This proposed mechanism is
90
applied to rationalize the observations of table 6-9 and table 6-10. In table 6-9, the lack of
side chains on azetidinone, sulbactam, and clavulanic acid results in the lack of
chemiluminescence; no a-proton on methicillin and cefotaxime results in no
chemilimiinescence; for penicillin V, the oxygen on the side chain will repels the attack
of 'O2 on an a-proton and the chemiluminescence is inhibited. In table 6-10, although
penicillin G emits the most intensified chemiluminescence, its chemiluminescent
intensity is far lower than luminol, lucigene, luciferin, and other chemiluminescent
compound. The conformation of penicillin G determines that the carbonyl and benzyl
moiety can not be on the same plane, and the lack of conjugated electron-delocalized
structure leads to decreased electron withdrawing ability, thus, the removal of the a-
proton is restricted. The variation of the acidity of a-protons also indicates that the
chemiluminescent intensities of amoxicillin < ampicillin < piperacillin < penicillin G,
which is consistent with their decreased electron-withdrawing abilities. Because 'O2 can
works as an oxidizer for the sulfur of cephalothin and as a base to hydrolyze the side
chain of hetacillin, whose five-member-ring side chain makes it more susceptible to
hydrolysis than that of the other antibiotics, it is not surprising that the chances for
chemiluminescence are decreased. The bulky side chain on the hetacillin can block the
attack of 'O2 on a-proton, which combined with ring strain facilitate the hydrolysis of the
side chain. In an attempt to explain the large chemiluminescent intensity obtained with
91
dicloxacillin, which does not have an a-proton, a particular mechanism is proposed in
which the hght is emitted by the dioxetan as shown below:
Extensive studies suggested that the most convenient method to produce
dioxetans and dioxetanones is following reactions of olefins with singlet oxygen [US-
IS 1]. Various dioxetans fall sharply into two classes. Dioxetans with alkyl aryl or other
substituents ("simple dioxetans") with little electron donating power yield triple exited
carbonyl products, whereas the second type, with strongly electron donating substituents
(mainly nitrogen and oxygen) gives very high yields of singlet excitation [132]. Possibly,
the dioxetan of dicloxacillin belongs to the second class and further investigations are
needed.
Finally, much data acquired fi-om the HPLC-MS, D^O, NMR, and TLC suggest
that the mechanism of chemiluminescence of p-lactam antibiotics is autoxidation initiated
by singlet oxygen.
92
Table 6-1: Effects of deuterium oxide on the intensities of chemiluminescence of
penicillin G.
Deuterium Oxide concn, %
Relative integrated chemiluminescent
intensity
100
70
50
204
61
22
20
Table 6-2: The response of spraying the three reagents on sample PGKO2 and sample
KO,.
PGKO2
KO2
color
# of spots
# of spots
Vanillin
brown
1 , L=3.0cm
none
lodine-Azide
yellow
1 , L=3.0cm
none
Ninhydrin
purple
3, L=1. 3, 2.5, 3.2cm
none
Table 6-3: The elution time of TLC developments.
Elution time (min.)
Average elution
time (min.)
Plate 1
Plate 2
Plate 3
Plate 4
Plate 5
Istelution
41
44
42
44
44
43
2nd elution
39
39
37
40
40
39
3rd elution
40
38
37
39
36
38
93
Table 6-4: The retention times of the reference compounds and sample PGKO2
Injection
Concentration
Retention Time (min)
Retention Time (min)
with UV at 254 nm
with PDA"
hydrogen peroxide
0.03%
1.63
1.692
3, 4-dihydroxyl benzoic acid
0.01 mg/ml
2.71
2.795
p-hydroxyl benzoic acid
0.01 mg/ml
4.19
4.192
2, 4-dihydroxyl benzoic acid
0.01 mg/ml
4.20
5.178
2, 3-dihydroxyl benzoic acid
0.01 mg/ml
4.41
5.033
p-hydroxyl phenyl acetic acid
0.01 mg/ml
6.28
6.805
benzaldehyde
1/250^
8.95
8.907
benzoic acid
0.01 mg/ml
9.00
8.754
phenyl acetic acid
0.01 mg/ml
13.60
14.464
salicylic acid
0.01 mg/ml
14.35
14.97
major peaks of sample PGKOj
2.0 mg/ml
3.61
4.267
14.40
14.433
21.99
25.333
^volumn ratio.
"on a different Exsil" ODS column.
94
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phase A = 0.1% acetic acid in 50:50
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acetate (pH5.72) + 0.1% acetonitrile in water
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95
Table 6-6: The products of recombination including dimerization after penicillin G
reacting with potassium superoxide.
OH
CH3 s-
/
-OH,
~S CH,
OH
296=149+149
dimer of Penicillamine
H3C-
H2N
/
-CH,
~S CH
OH
312=296+16
^ — ( )— chj:h3 nh,
0=^ SH3C
OH ■' " ■
632=296+354(352+2H)-H20
352=Benzylpenicilloic Acid
642=308(Benzylpeniltoic Acid)+
334(Benzylpenicillenic Acid)
Mechanism:
RSH + B-
RS- + O2
RS- + 02-
2RS
BH + Oi^
RS- + BH
RS + Oj-
RS + 02^
-*- RSSR
-.► OH- + B- + O2
96
Table 6-7: The major products of hydrolysis of penicillin G after interacting with
potassium superoxide.
Mobile Phue A
[0.1%HOAcin 50 50H2OACN]
MobilePhiseB
P 01 M N»IOAc(pH 572) +01% ACN]
Compound
RT7.79 min
RT19.94min
benzylpenicilloic acid
C.jHaoNAS, MW352
[M+H]*=in/z 353.4
RT9.58 min
RT20.08 min
benzylpenillic acid
C,6H,8N204S, MW334
[M+H]*=m/z 335.4
HO — f^
\..-^ — / CH
H,
■CH,
Qr <^
RT 10.09 min
RT20.91 min
ben2ylpenicillenic acid
Ci6H,8N204S,MW334
[M+Hr = m/z 335.4
RTl 1.77 min
RT22.15min
penicillin G
Ci6H,8N204S,MW334 ">"=
[M+Hr=m/z335.4
RT12.26min
RT22.59 min
benzylpenilloic acid
C15H20N2O3S, MW308
[M+H]+ = m/z 309.4
/ •■^-
97
Table 6-8: The major products of oxidation of penicillin G after interacting with
potassium superoxide.
Mobile Phase A
[0.1%HOAc in 50:50 H20:ACN]
Mobile Phase B
(O.OIM NH40Ac(pH5.72) + 0.1%ACN1
Compound
RT11.54min
RT20.74 min
benzylpenilloic acid sulfoxide
C15H20N2O4S, MW324
[M+H]*= nVz325.40
RT11.57tnin
RT20.56 min
*
.0
HO^
Y^N~^ CH3
benzylpenillic acid sulfoxide
C,6H,J^20sS, MW350
[M+H]* = m/z351.39
98
Table 6-9: The six p-lactam antibiotics that emit no chemiluminescence.
O
%ll
HN-
O
/
HO V O^
\
o
azetidinone
MW71.08
C3H5NO
sulbactam
MW233.24
CgHuNOsS
clavulanic acid
MW185.18
C8HnN04
H,C
HjC.
H,C'
,NH ^^^3
^^
„^
V
OH
methicillin
MW3 80.41
CnH^oNiOsS
Penicillin V
MW350.39
C.sH.sNjOsS
"Y
HN
°/
OH HjC
cefotaxime
MW455.46
CsHnNsOySz
^
NH,
99
Table 6-10: The seven P-lactam antibiotics that emit chemiluminescences.
Antibiotic
Structure
Relative Intensity
Penicillin G
°<)M
100
Dicloxacillin
:?p^-
54.4
Piperacillin
o
47.9
Anipicillin
43.3
Cephalothin
"T^^
8.3
Amoxicillin
OH
5.6
Hetacillin
OH
0.6
100
TO
U]
o
ux..
10
20
Time (s)
Figure 6-1: The profile of chemiluminescences of penicillin G in different solutions with
varied deuterium oxide to water ratios.
101
1=
15 i
o
Q
a
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O
o
90ft
SZC'I.
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102
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107
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108
I6»-99^^
109
r~^
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1;
Pk
no
Ill
57 mm
43 mm
After the third run ^'^'^"^
28 mm
and spraying
22 mm
6 mm
I
covered
by paper
100 mg/ml of sample PGKO2
solution 3(y)
solution 2(y)
solution l(p)
After the first run
and spraying
47 mm
.
19 mm
•
12 mm
•
4mm
•
solution 3(b)
solution 13(p)
solution I2(p)
solution ll(p)
C: solution 1
D: solution 2
E: solution 3
p: purple
b: brown
Figure 6-12: The schematic diagrams of TLC assays on sample PG and sample PGKO2
112
55 mm(y)
The first run, and spraying
(1) ninhydrin, (2) iodine-azide 21 mm(y)
6 mm(p)
The second run
and spraying
62 mm(y)
58 mm(y)
58 mm(y)
■■"-
''-"'
53 mm(y)
46 mm(y)
' -' 'I'
53 mm{y)
46 mm(y)
50 mm(y)
-''-'■ ''-"-'
40mra(p)
»
38 mm(y)
■'. -' '' '
—
The third run
^
30 mm(p)
and spraying
23 mm(p)
19nim(p)
t
- -
16 mm(p)
•
solvent front
44 mm(y)
21 mm(y)
58 mm(y)
55 mm(y)
50 mm(y)
38 mm(y)
A: 10 mg/ml of sample PGKO2 p: purple
B: 10 mg/ml of sample PG y: yellow ■/
Figure 6-13: the schematic diagrams of preparing the samples for mass spectroscopy by
the TLC separations.
113
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r> rs oj r\ w rs o&
« ■-• rj ro ** *^ 00 «^ o^ ^
.u. .VH-incotOftjmooojffs
:i£ <roc
— w cu M ro if> ■«• »-
TT-r
;si
Figure 6-15: The chromatograms of sample PG (a) and sample PGKO2 (b). The buffer
system: pH5.66 phosphate buffer containing 0.1% acetonitrile.
115
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Figure 6-16: Schematic of major processes occurring in electrospray.
116
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118
Compound 350b
MW350
Oxidation
Benzylpenicillenic Acid
MW334
RT20.91 mia
Benzylpenicilloic Acid
MW352
RT 19.92 mia
Oxidation
Compound 368
MW368
Oxidation
Penicillin G
MW334
RT22.15min.
BenzylpeniUic Acid
MW334
RT20.08
Benzylpenilloic Acid
MW308
RT22.59 mia
Oxidation
P..
o
Compound 324
MW324
RT20.81
Compound 350c
MW350
Oxidation
Compound 350a
MW350
RT20.08 mia
Compound 350b, 350c, 368 are detected by different HPLC gradient.
Figure 6-19: The scheme of generation of sulfoxides after penicillin G reacts with
potassium superoxide.
119
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z
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l' I
CHAPTER 7
SUMMARY
The conversion mechanism of superoxide anion in aqueous system
can be described as the folio wings:
Superoxide dismutation: SOi' + 2H* »- H2O2 + OiCAg)
Haber- Weiss Reactions: H2O2 + 02" — ^ OzCAg) + OH" + OH
H2O2 + OH .► H2O + H02-
H02- ^ H" + 02-
OH + O2- ^ 02('4) + OH
Quenching reaction: 02('Ag) + 02" ^ ^Oj + Oj" + 22 Iccal
Chemiluminescent reaction: 02('Ag) *- ^02 + hY(1270nm)
U + H2O ^ 2H2O2
The superoxide anion has been proved to be relatively unreactive toward most
biological components. This is supported by the observations that no chemiluminescence
120
121
was detected when superoxide anion in 18-crown-6-ether-acetonitrile was used to oxidize
penicillin G in acetonitrile, methanol, butanol, and chloroform. In these solvents
superoxide anion will not readily convert into other reactive forms and these observations
indicate that superoxide anion itself is not capable of oxidizing penicillin G with the
emission of chemiluminescence. However, in aqueous solution, through the superoxide
dismutation reaction, superoxide can generate reactive species, for example, hydrogen
peroxide and singlet oxygen ('O2). Hydrogen peroxide is used extensively as a source of
^Oj and it is not reactive enough to oxidize the thirteen P-lactam antibiotics with the
resultant emissions of chemiluminescence. This leaves 'O2 as the most probably reactant
for the oxidation of penicillin G and the emission of chemiluminescence. The
involvement of 'O2 in the chemiluminescent reaction of penicillin G after reacting with
potassium superoxide was strongly indicated by the D2O experiment using deuterium
oxide instead of water as the solvent for penicillin G. Meanwhile, no chemiluminescence
was measiu-ed with penicillin G in phosphate buffer. It is believed that 'Oj is probably
quenched by phosphate buffers. The experiments had indicated that pH4.5-7 is the best
pH range for the chemiluminescences of p-lactam antibiotics because they are stable in
the solutions with pH values close to neutral and the superoxide is easily converted into
'O2 by superoxide dismutation reaction. The degradation studies of penicillin G by
HPLC/ESI(or APCI)-MS/MS suggested that the chemiluminescence of |3-lactam
antibiotics was generated by the autoxidation initiated by 'Oj. This type of reactions is
122
also the pattern of luciferins and many related compounds. The maximum wavelength at
535 nm of chemiluminescent penicillin G is very close to that of Lucifer's at 562 nm. The
low efficiency of penicillin G's chemiluminescence is consistent with its chemical
structure and the fact that superoxide is a highly efficient 'Oj quencher. The presence of
'O2 was also proved by the characteristic 1268 nm chemiluminescent emission spectrum
of 'O2 as discussed in chapter 1 . On the contrary, no detectable hydroxyl radical was
involved in the degradation of penicillin G, because no aromatic hydroxyl substituted
derivatives was observed by NMR and mass spectrometry. Perhaps, the hydroxyl radical
can be postulated to be a short-lived transient intermediate, which is produced by the
Haber- Weiss reaction, however, the data collected in this dissertation reinforce that 'Oj
rather than hydroxyl radical is the prime oxidant in the reaction system. Even though
transition metals are available in vivo, the metal-catalyzed Haber- Weiss reaction will face
competition from Fenton-type reactions, in which H2O2 interacts with transition metals or
metal complexes to generate hydroxyl radicals. Indeed, The questions arise which is the
crucial toxic species, singlet oxygen or the hydroxyl radical, and whether the hydroxyl
radical comes from the metal catalyzed Haber- Weiss reaction or from a Fenton-type
reaction. The answer to these questions must be specified in order to better understand
oxygen toxicity and develop new therapeutic strategies as well as new antioxidants.
»-*.?■.
W
GLOSSARY
APCI Atmospheric pressure chemical ionization
APT Attached proton test
ATP Adenosine triphosphate
BHT Butylated hydroxytoluene
CFFAB Continuous-flow fast atom bombardment ionization
CI Chemical ionization
CNS Central nervous system
DEPT Distortionless enhancement by polarization transfer
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
EI Electron impact
ESI Electospray ionization -
ET Electron transfer (or exited state)
FAB Fast atom bombardment
FWHM Full width at half maximum
GC Gas chromatography
HPLC High pressure (or performance) liquid chromatography
HV High voltage
MS Mass spectrometry
NMR Nuclear magnetic resonance
ODS Octadecylsilane
PB Particle beam
PDA Photodiode array
PMT Photomultiplier tube
RSP Relative standard deviation
SOD Superoxide dismutation
TLC Thin-layer chromatography
TME Tetramethylene
TSP 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (or thermospray
ionization)
UV Ultraviolet
123
APPENDIX A
THE PRODUCTS OF HYDROLYSIS OF PENICILLIN G FOLLOWING THE
INTERACTION WITH POTASSIUM SUPEROXIDE - THE CHROMATOGRAMS
AND MASS SPECTRA OF HPLC/ESI-MS/MS.
, ..J'
o
-<M
n
O
-o^
^
cf^
-1^
e^;t2
0— p=
l:
to
5
to
55
0>CO
"5-yjf
■on.
to to f
So
5 ml
o™
SI
!Kh
1 1 Tllllllllllllll
00 to rt C4
83uepunqv 8Ai;e|au
1
°i
aoii_
Kh
1 T T-n I 1 I I [ I I I I I I 1
o o o o o
CO <D ^ OJ
douepunqv OAiieis^
— T 1 T— — I 1 1 f-
e S 8 S S S 2
'B
a,
o
00
00
>
o
CI,
u
125
126
308 - RT12.26 min - HPLC / ( + ) ESI - MS / MS
(XX,:
'-YX-
C,4H„N20S
MW262.37
[M+H]* = 263.38
^O^-^h;
C8H9NO
MW135.16
[M+H]*= 136.17
k.-^'^^./^.H
rx-
CsHjoNjOsS
MW308.39
\M+HY = 309.40
C4H7NS
MW101.17
[M+H]*= 102.17
*-\
C5H5NOS
MW127.16
[M+H]*= 128.17
C,5H„N202S
MW290.38
[M+H]* = 291.39
HjN*
.CH,
CHmNzOzS
MWl 90.26
[M+H]*= 191.27
HjCi
^^
CtHiiNOzS
MW173.27
[M+H]* = 174.28
CjHiiNOS
MW145.22
[M+H]*= 146.23
Figure A-2: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic acid.
127
o
o
m
6.
O
C3
O
O
^-' ns
ex
It-I
o
CO
00
>
u
d
u
H
t
<
i
•I-H
fa
128
308 - RT12.22 min - HPLC / ( - ) ESI - MS / MS
(^
H,
C15H20N2O3S ho'
MW308.39
[M-H]- = 307.39
H,
C,4H,8N20S
MW262.37
[M-H]- = 261.36
^
CuHhNzOS
MW234.32
[M-H]- = 233.31
CnHioNjOS
MW230.28
[M-H]- = 229.28
Figure A-4: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilloic
acid.
129
;:\LCQ\data\Spec\seq1924f 09/12/97 04 0304
m phase C18 HPLC/ESI-MS
>#: 809-810 RT.iiM-iiM A\/: i ^5 6: S i2 00-i2.62 22 34-22 39 NL
r: ♦ c ms ( 75.00 - 1200.00]
Penicillin G [8/28/97. Sun]
352'f-CM + NH+]*
il'vl;-'
685
3W + 335
587 <-!
^1
K-\ 45g.476i96 569 64,2 g^9|| ,_IP6 ^^^ 335 917 995 1026 1Q61 1117 1140
■»00 500 6(1)0 ' ' ' ' 7(1)0 ' ■ ' 'edio' 900 1000 ' 'n'oo' ','■>',
iT. 19.35-24.70
100
1100
1200
22.13
8 0^
S100-,
C
I 50^
>
a
Sioo^
50-^
NL-
1.16e7
BaMPeak
20 06 / \ '"'^'
19.62 ^^^^ 20,5 20^ .00 ,,,, ,,,, / V ,,^ ^^^ ^^^^ ^3^ ^3^^ ^^^ ^^^^^ ^^^^^ ^^^ ^ -—
"lis ^^ ^ NL:
2.02E7
mfta
334.S-335.S+
20 06 / \ 351.5-352.5
19.62 19,89 ^ 20.35 20.55 20,93 2^,07 21.56 21.81 / V^ 2Z43 22.68 23^ 23.42 23.55 24.10 24.32 24 59
22?I5 NL
1.81 E6
m/2«
308.6-309.6-*
19.92
20.87
19.76 .' \ jO^OS 20 35
.-^
4i:??.ii35 21,65 2K93
100
22.59
22.11"
V22.79 23,13 J^^Jk^
,2jj6 24.12 24.50
19.5
20.0
20.5
20.39
21.0
I 22,24
^MLJMl^W 20,48 20.73X21.17 21.56 ''5ili^!^ " ,22.84 23.17 2^33 23. 70 24.OI 24.41
r.
2<.5
22.0
Time (min)
22.5
23.0
23.5
352.6-353.6
NL:
8.43E5
m/z=
350.6-351.6*
367.6-368.6
24.5
C:M.CQ\data\Spec\seq 1 924v
rev phase C18 HPLC/ESI-MS/MS
!>#:S31-537 Kl : 11.77-11.90 AV- ? HL- 3 lyg'
T: ♦ c Full ms2 335.10 [ 90.00 - 400.00]
160
100-
^ 80-;
c 60-
§
40-
■
a. 20-
-
114
qJ
.
100 120
15a
176
140 160
180
09/13/97 02:12:26
PenicJIIIn G
307 335
2<i0 • 2i0- ■ '2^0'' 2d0- ■ '2^0 '3io' ' ' 3^0 ' ' ' 3I0 '^ ' 3^0 ' ' '3^0
40O
Figure A-5: The positive HPLC/ESI-MS/MS of penicillin G and MS/MS of m/z 335.
130
\LCQ\dala\Spec\seq 1 924M
V phase C18 HPLCyESI-MS
*: 501 RT: 11. 8S AV: 1 SB: 7i 4.53-5.05 NL 9.2dE5
- c Full m$ [ 75.00 - 800.00]
lOOj
i 60^
I 50^ 119
p
10^
09/12/97 18:58:26
Penicillin G
83
m
157 192
289
331
667
^M9
J50 390
4p9
3^«^1A15 458
639
2'<333tl
558
683
<6o Uio ' 560' '5io' ' ' 'eio' ' ' 6io' '71
701
'700'
P7 742 258
' , ■■■■■ , [ , ', .
100
250
360
350
m/z
7io ' sio
T: O.O1 - 30.03
100
50^
I "
|100t
»
I "
- lOOn
50^
100
50-
o-l— .
11.89
0.00
0.23
00 '-'^
^0,00
2.92 3.70 4.91
0.00 0.00 0.00
6.52 7.14
.00 fl.00
O.M^
^n°;i^ "i^ro^fi? ";" 1i£? 16.31 17.41
.00 n
.00 ojDO ^00 oQQ 0,00 00
20.47 21.24 22.57 24.51
0.00
27J1 28.43
O.Qp OjO_
11.89
333.44
10.22
333.03
12.15
333.04
10.07
307.04
12.39
307.06
13.22
307.02
11.91
351.16
jS!L.hiii
10
10.98
351.00 n
12
-»•■■■•■ .r
14 16
Trme (min)
" I '
18
1 ' •
20
•r I
22
IT
^
IT
NU
5.31 E«
BasePeali
miz'
7$.M00.0
NU
a.30ES
333.0-334.0
2.68E4
m/z»
307.M08.0
NL;
4.12E4
351.0-352.0
30
:::\LCQ\dala\Spec\seq 1 924w
ev phase C18 H PLC/ESI-MSA^S
5* 527-534 m 1169-11.84 AV: 8 NL- 9 73E4
r: - c Full ms2 333. 10 ( 90.00 - 400.00J
100
8 80-
c "•
m
I 60
m
K 20-
120
09/13/97 18:45:54
192
180
2<lo' ' '2io'
2^0 ■ '2^0'
m/z
Penicillin G
289
288
2^0 'sio'
320
ur
3io '3^0'
Figure A-6: The negative of HPLC/ESI-MS/MS of penicillin G and MS/MS of m/z 333.
131
Penicillin G - RT22.15min - HPLC / ( + ) ESI - MS/MS
C,6H„NAS '
MW334.39
[M+H]* = 335.40
^^=0
C,oH,N02
MW175.18
[M+H]^= 176.19
CsHsNOzS
MW159.20
[M+Hr= 160.21
HjC
CHjNS
nVzl 14.18
Penicillin G - RTl 1.89 min - HPLC / ( - ) ESI - MS/MS
H0~^
CsHlNAS
MW334.39
[M-H]- = 333.38
CjHtO
m/zl 19.14
■s><x(.
C,5H„N202S
MW290.36
[M-H]- = 289.35
^ Ovh-
C„H,5N02
MW193.25
[M-H]= 192.24
Figure A-7: The interpretations of the positive and negative HPLC/ESI-MS/MS of
penicillin G. * ^
ri..
132
8f
TTTT
o
1 1 1 1 1 1 1 1 1 1 1 1 r [ 1 1 ) 1 1 1 [ p 1 1 m 1 1 [ 1 1
o o o o o o o
CO h- (O U> Tt fO CVI
30UBpunqv aAjjeiay
CO
1 " " I " " I " "I "" 1 < " 1 I M I I I I
S 9 2 o Q o o
o OT CD r~ <o tn ■«
aDuepunqy 3Ar)e|a^
o
I
I— (
CO
O
>
CO
O
D,
(U
S3
H
do
<
I
133
334a - RT20.33 min - HPLC / ( + ) ESI - MS / MS
r
CsHi^NzOjS
MW3 16.37
[M+Hr = 317.38
C16H18N2O4S
MW334.39
[M+H]* = 335.40
o
C,HuN04S
MW2 17.24
[M+Hr = 218.25
.0 T
,CH,
.. ■ "^
HN*=
CyHnNOaS
MW173.23
[M+H]*= 174.24
C6H9NO2S
MW159.20
[M+Hr= 160.21
.0
.CH,
CH,
r^
CisHijNaOzS
MW290.38
[M+Hr = 291.39
C5H,NS
MW115.19
[M+Hr= 116.20
Figure A-9: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenillic acid.
,f:
134
E
o
O
a.
o
o
o
CO
0)f
5^
LU
CO o^
WI
raZ
Is
o 2
CM
UJ
Q
Sl
-L-
Sf"
- $2f
Mnip
I < I " ■ 1 1 " " I " " I " " I "
o o o o o
03 r- <D IT) -^
o
-CM
CM
> — o
iuOLEiioo
i^'^ J= =><00<M
10
o
1$
CM\.
CM
u
81-
o o o o
CO CM •>-
k/)u.
oouepunqv aAijeiay
i""i""i""i""A""A""A""i""i'
0<7)<Ot^u5i5-«(OCM
o
CSJ
o
ID
E -?
93uepunqv wiiieiaij
o
C/3
C/D
00
W
u
>
• i-H
CO
O
D.
(U
135
334b - RT21.51 min - HPLC / ( + ) ESI - MS / MS
HS — \ — ^^
C,6H„N204S
MW334.39
[M+H]* = 335.40
CsH.sN^O^S
MW288.36
[M+H]* = 289.37
Q\
CHioNzOj
MW202.21
[M+Hr = 203.22
HS — 1 — '-"s
HjC;
-Y
CsH.oNOzS
m/zl60.21
\ /
■oAo
C,Ji,N02
MW175.19
[M+Hr= 176.19
HO
H,C NH*
^ //
MW127.14
[M+Hr= 128.15
CsHi.NO
MW113.16
[M+Hr= 114.17
Figure A-1 1 : The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicillenic
acid.
136
-s
o
-eg
gf
ino
..o
1^
cnco ,
■&iy !
Q)0 (
i2-J
oOl
Bo
■§'®F
8ft
I Ml 1 I I I 1 r I 1 r-T-pi
o o o o
00 to -Tt Csl
aouepunqv aAijeiay
souepunqv 3Ai)e|3H
i?H
15
2<^
^^
"^1
3
a
«» ■
IB -
oi '
o
"«
8,
¥^T'
o
C3
o
c
o
00
00
U
u
_>
*■♦-»
a
u
H
137
334b - RT10.09 min - HPLC / ( - ) ESI - MS / MS
C,6H,8N204S
MW334.39
[M-H]- = 333.38
*> f .
:"v«._ ?^"5 :.
;* . '^
\'"'- ;"%<;
C.sH.gNjOjS
MW290.38
[M-H]- = 289.37
C,5H,6N202S
MW256.30
[M-H]- = 255.29
Figure A-13: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenicillenic
acid.
138
o
o
'B
'o
•a
u
ex
u
x>
o
00
C/3
00
u
aouepunqv aAiieja^
'■4-»
O
a
H
139
352 - RT19.49 min - HPLC / ( + ) - ESI - MS / MS
CH,
CsHjoN^OjS
MW308.39
[M+H]* = 309.40
C,3HhN202S
MW262.33
[M+Hr = 263.33
CsHzoNzOjS
MW352.40
[M+H]*= 353.41
CjH|,N04S
MW2 17.24
[M+Hr = 218.25
C6H9NO2S
MW159.20
[M+Hr= 160.21
C,6H,8N204S
MW334.39
[M+H]* = 335.40
C,H„N02S
MW173.23
[M+Hr= 174.24
n4
(
s
CsHsNS
m/zll4.18
Figure A- 15: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicilloic
acid.
140
o>
B
O
o
Q.
iii
a.
X
00
O
SI
^
(DO
(QI
raZ
ro
Ora
9^
C4
H
CM
lO
5°
CDO
O
W
E
ym I imifiTi I inTrprT
o O o o o
o o) CO r^ <o
lyrrrrTrrrrprnjTTnjnTi
o o o o o
lO Tj- CO oi ■•-
o
-<D N
'1
90uepunqv aA{)e|3^
QJ u_ E*-oo
O
o
O
•a
— : • s
33uepunqv 8At)e|3^
X>
o
00
00
I— I
00
u
-1
c
H
i
141
352 - RT18.36 min - HPLC / ( - ) ESI - MS / MS
C,5H,5NO,S
MW289.35
[M-H]= 288.34
A
C13H12N02S
MW245.29
[M-H]- = 244.29
CHijNAS
MW260.31
[M-H]- = 259.30
MW352.40
[M-H]- = 35 1.39
C.sHjoNjOjS
MW308.39
[M-H]- = 307.39
/
CsH^N^O^S
MW170.18
[M-H]- =169.18
C,4H,J^203S
MW292.35
[M-H]- = 291.34
r^
CsH.jNjOjS
MW2 16.25
[M-H]- = 215.25
C,2H,4N20S
MW234.23
[M-H]- = 233.31
C„H,oHO,S
MW274.29
[M-H]- = 273.28
Figure A-17: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenicilloic
acid. . ■•
'.'> ** i> '>
U *
APPENDIX B
THE PRODUCTS OF OXIDATION OF PENICILLIN G FOLLOWING THE
INTERACTION WITH POTASSIUM SUPEROXIDE - THE CHROMATOGRAMS
AND MASS SPECTRA OF HPLC/ESI-MS/MS.
i: T ^
vr;,--^
o
C3
^'
CO
! a>
: to
CI
O)
0)
So
CO o
So
tx-<r
cox
roZ
li
8f
§/
CD<N
1 1 1 1 1 M 1 1 1 1 1 1 n 1 1
O 2
It ..
Hjl IIM I II || II II I II I 1)1 I
~ o o
CO CM
to lO
TJTT7T
r<^
■f
+ <N —
"O .. "o • O
lu u, Eooo
J^ObW^o
o
2 V- 1— U-rjoO"*
-o
.^
souepunqv 9Aj)e|a^
r^ © m
&,
o
u
X
<B
3
(/I
u
■♦-»
tt-l
o
t/3
00
C/3
U
H-l
>
o
cx
eouepunqv SAiieis^j
IX,
143
144
312 - RT28.16 min - HPLC - ( + ) ESI - MS / MS
HsCv^^H^
HjN*
OH
C5H7NO2
MW113.il
[M+Hr= 114.12
H3C
C4H„NOS2
MWl 53.26
[M+H]*= 154.26
H,C
CoH.sNjO^Si
MW294.38
[M+H]* = 295.39
C5H6NOS2
ni/zl60.26
OH
Hjn:
CH3 \
/
-S
-CH,
CH,
H^ir-^^
CoHjoNjOsSa
MW3 12.40
[M+Hr = 313.40
C^HgNzOzSz
MW204.26
[M+H]* = 205.27
'SH
H2N*
C2H5NS2
MWl 07. 19
[M+Hr= 108.20
H3N*
OH
H,C-
O
CH3 \
/
-S
OH
QH,6N205S2
MW284.34
[M+H]" = 285.35
Figure B-2: The interpretation of the positive HPLC/ESI-MS/MS of the sulfoxide of
penicillamine dimer.
...?.•/'■ •''..' ,\.r.
145
o
rt^
■*
•* .. i9o • o
j«"3;^Sa
o
Zm 1-u.nco-*
souepunqv aA{)e|3y
^CV.
aouepunqv 9A!}et3^
U
O
u
O
00
00
00
u
PL,
>
<2
•t-4
{X4
146
312-RT28.il min-HPLC/(-)ESI-MS/MS
CH3\
H3C-
HjN
-CH,
CH,
OH
CoHioNzOsSz
MW3 12.40
[M-H]-=311.39
N
./=\.
% ^'
CH3
C6H5NOS2
MW171.23
[M-H]-= 170.22
H3C-
HjN
CH3\-
/
-s
-CH,
CH,
^NH,
C9H20N2O3S2
MW268.39
[M-H]- = 267.38
Figure B-4: The interpretation of the negative HPLC/ESI-MS/MS of the sulfoxide of
penicillamine dimer.
147
aouepunqv 8Ai)e|9^
o
"a
O
C/3
I
pq
U
>
CO
O
O.
(L>
fS
I
CQ
(3JQ
148
324 RT21.83 min - HPLC / ( + ) ESI - MS / MS
CHlNjOsS
MW306.38
[M+H]* = 307.39
C,4H„N202S
MW278.37
[M+H]* = 279.38
ChHzzN^O
MW234.34
[M+H]* =235.35
CsHiiNOjS
MW177.22
[M+Hf =178.22
C,5H2oN204S
MW324.39
[M+H]" = 325.40
"!<«,
K
NHJ
CHnNzOzS
MW 188.24
[M+Hr= 189.25
C9H9NO
MW147.18
[M+Hr= 148.18
CsHioNaOS
MWl 58.22
[M+Hr= 159.23
"^
C4H6N2OS
MW130.16
[M+Hr= 131.17
Figure B-6: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic acid
sulfoxide.
149
•i-H
X
O
'o
1
a,
o
o
CO
00
I
00
W
U
h-1
>
u
d
u
XI
H
m
aouepunqv dAiieia^
150
324 - RT21.80 min - HPLC / ( - ) ESI - MS / MS
C15H20N2O4S
MW324.39
[M-H]-= 323.38
°\ CH,
"w^^"'
HH-^
C,4H2oN202S
MW280.38
[M-H]- = 279.38
C,3Hi4N202S
MW262.33
[M-H]- =261.32
C5H5NO3S
MW159.16
[M-H]-= 158.15
CsH.sNiO,
MW274.32
[M-H]- =273.31
C14H22N2O
MW234.34
[M-H]- = 233.33
O N=CH,
M
O- CH,
C4H5NO2
MW99.09
[M-H]- = 98.08
C,4H„N20
MW230.31
[M-H]- = 229.30
Figure B-8: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilloic acid
sulfoxide.
151
9
CM
O
to
n
«o
-
n
CO
h-
SL
▼*
O
°
fO
h- c3L
-
aouepunqv a/uieiay
=?feo
loSca
CO 3O
y-
„u.°
i^
^1^2.
^
I5J
■cHj''
q CD ,--
^ 00 ^^-=-
■» l"iM"ii
SDuepunqv 3A|)e|3)j
152
350a - RT22.35 min - HPLC / ( + ) ESI - MS / MS
^{
C,6H,6N204S
MW332.37
[M+H]* = 333.38
C.iHioNzOa
MW202.21
[M+H]" = 203.22
"~0
C^HhNjOsS
MW322.33
[M+H]* = 323.34
CsHuNjCS
MW350.39
[M+Hr = 351.39
CiHnNzOsS
m/z305.37
CHioNzOj
MW260.33
[M+Hr = 261.34
^
CHijNOjS
MW235.25
[M+H]^ = 236.26
C4H5NOS
MW115.15
[M+H]+= 116.16
C,6H,7N204
m/z301.32
1
r
^
^ir-
0>X"
C6H9NO3S
MW175.20
C,5H,5N204
[M+H]*
= 176.21
ni/z287.29
V
"V— «H.
'\ 1
J
ivr-i
C6H,N02S
CH^NOjS
MWl 59.20
MW147.19
[M+Hr= 160.21
[M+Hr= 148.20
Figure B-10
sulfoxide
': The interpretation of the positive HPLC/ESI-MS/MS of benzylpenillic acid
153
o
CD
OL
(0
lU
OT
i:!'0
rn <»
->
u«
UI •
W o
r><
a)C1
&■*
WT
rar
-o «
flfo
S-
o £
5°
to
«
_ E
cn =
oo o
» .'.
t/)u.
o
in
o-
n
^
o
-co
SO
CM
gr:
3^
u I ri [ I I 1 I I I [ I r I I I I I I I I I I I I I I I 1 1 I I n I [ I I
oooooooo
0OI-<DIOV<O(Mt-
aouepunqv 3Ane|32j
Jo O = oj9d
^
___i
^ O
N O
1 ' ' ' ' I " ' ' M ' • ' I I I I ' [ I I I 1 J 1 I I T I IT
■^ S S S o o o
- (O in n CO eg *-
93uepunqv OAiieia^
"CO
•s
•i-H
X
^
3
l/J
-o
o
"CO
rt
o
V.
'«^
u
c
a.
h
o
ICi
E
H
■cm'^
o
^
^
o
.00
t/)
v:
^
S
HH
C/)
^
.(D
u
J
a
a
(D
>
•4—*
rt
<30
S
(I)
a
<u
^
sa
H
^
'—>
ffl
CNJ
(U
IX,
154
350a - RT22.38 min - HPLC / ( - ) ESI - MS / MS
\
H3
CHnNOjS
MW191.24
[M-H]-= 190.24
C,6H,6N204S
MW332.37
[M-H]- = 331.36
C,5H,6N202S
MW288.36
[M-H]- = 287.35
CH,
C^H.gN^OsS
MW350.39
[M+Hr = 349.38
C11H15NOS
MW209.31
[M-H]- = 208.30
C,5H,8N203S
MW306.38
[M-H]- = 305.37
CH.sNOjS
MW241.30
[M-H]- = 240.30
CK^"
CH,
C,5H,5N,02
MW256.30
[M-H]- = 255.29
CoHisNOS
MW197.29
[M-H]- = 196.29
Figure B-12: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilUc acid
sulfoxide.
i .
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156
350b - RT24.13 min - HPLC / ( + ) ESI - MS / MS
CsHisNiOiS
MW332.37
[M+H]* = 333.38
O^
Ci,H,6Nj04
MW232.2g
[M+H]* = 233.29
M_/y^^
C„H,oN202
MW202.21
[M+H]* = 203.22
Cl4HMN20iS
MW322.33
[M+H]* = 323 34
v.
C,5H,6N20,S
MW304.36
[M+H]* = 305.37
C,6H,^20iS
MW350.39
[M+H]* = 351.39
HO
HjC nh;
CsHjNOj
MW115.13
[M+H]* =116.14
CHijNOsS
MW235.25
[M+H]* = 236.26
3
»-""i
•n
CeHsNOiS
MW175.20
[M+H]* =176.21
rT"
CsHaNjO.
MW260.25
[M+H]* = 261.26
«\
CsHsNOjS
MW14719
[M+H]* = 148.20
CsHjNOjS
MW159.16
[M+H]* =160.17
C5H7O2S
m/zl31.17
CsHisNiO.
MW300.31
[M+H]*= 301.32
C,5H,4NA
MW286.28
[M+H]* =287.29
Figure B-14: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicillenic
acid sulfoxide.
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it .'. aouepunqv sAijeisy
^ aouepunqv 9/uiB|9a
c;
158
350b - RT24.24 min - HPLC / ( - ) ESI - MS / MS
s — O'
O^H,
H,C
C,5H„N:03S
MW306.38
[M-H]- = 305.37
C,4H,2N20jS
MW288.32
[M-H]- = 287.31
C,6H,6N204S
MW332.37
[M-H]- = 331.36
OH HN
O- CHj
S-
HN
C7H,3N03S
MW191.24
[M-H]-= 190.24
\ //
CH.gNjOsS
MW350.39
[M-H]- = 349.38
HX
%-V"'
C8H7N03S
MW197.21
[M-H]- = 196.20
C,4H,oN203
MW254.24
[M-H]- = 253.24
OH
C6H„N05S
MW209.21
[M-H]- = 208.21
Figure B-16: The interpretation of negative HPLC/ESI-MS/MS of benzylpenicillenic acid
sulfoxide.
159
a
CO
o
Pi
•a
O,
Cm
o
00
00
I
00
u
douepunqv SAiieid^
*■*-»
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w
O
u
H
oq
160
350c - RT35.74 min - HPLC / ( + ) ESI - MS / MS
C,4H,4N205S
MW322.33
[M+H]* = 323.34
n HN--\
" 6
C,5HnN203S
iii/z305.34
"^^:
CisHnNiOj
MW268.27
[M+H]^ = 269.28
C,4H,6N202
MW244.29
[M+H]* = 245.30
C8H,2N204S
MW232.25
[M+Hr = 233.26
C,6H„N205S
MW350.39
[M+Hr = 351.39
C4H5O2S
m/zl 17.14
- :::>a/
t
o
C8H9N04S
MW2 15.22
[M+Hr = 216.23
CgH,NO
MW135.16
[M+Hr= 136.17
CsHtNOsS
MW197.21
[M+H]"= 198.21
Figure B-18: The interpretation of the positive HPLC/ESI-MS/MS of peniciUin G
sulfoxide.
■Q -.^
:x'-^
161
aouepunqv SAjieiay
Z(0 h-U-COO)-*
«
'2
CO
o
1=1
'S
o
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t— I
00
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>
M
U
OS
I
PQ
9J
tM if
ODUepunqv 8Ar)e|3u
IX,
162
350c - RT35.95 min - HPLC / ( - ) ESI - MS / MS
C,6H2oN204
MW304.34
[M-H]- = 303.34
C,5H,8N204
MW290.32
[M-H]- = 289.31
C,4H,4N204
MW274.27
[M-H]- = 273.27
C.sHigNzOjS
MW350.39
[M-H]- = 349.38
CsH.eNjOjS
MW336.36
[M-H]- = 335.35
C|5H,4N204S
MW3 18.34
[M-H]- = 317.34
C,3H,5N03S
MW265.33
[M-H]- = 264.32
C8H,2N204S
MW232.25
[M-H]- = 23 1.24
o-
QHiiNOzS
MW173.23
[M-H]- = 172.22
Figure B-20: The interpretation of the negative HPLC/ESI-MS/MS of penicilUn G
sulfoxide.
-., f ■
163
O
o
t
o
o
o
00
00
I
GO
u
aouepunqv SAne^a^
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w
O
I
00
164
366a - RT21.62 min / HPLC - ( + ) ESI - MS/MS
V° H.
Ni-y^
C16H18N206S
MW366.39
[M+H]^ = 367.39
\
C16H16N205S
MW348.37
[M+H]" = 349.38
C10H7NO2
MW173.17
[M+Hr= 174.18
C15H18N204S
MW322.38
[M+H]* = 323.38
+
OH
C6H11N04S
MW193.22
[M+H]*= 194.22
H,C NH
H3C ^ — OH
O
C6H9N02
MW127.14
[M+Hr= 128.15
Figure B-22: The interpretation of the positive HPLC/ESI-MS/MS of compound 366a-
penicillin G sulfone.
.i * V
•d
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165
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el
a
w
O
•a
ex
o
s
o
• ^^
I
VO
VO
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1/3
00
00
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h-1
douepunqv BAije|8y
>
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a,
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H
en
I
CQ
i
166
366b - RT22.71 min / HPLC - ( + ) ESI - MS / MS
HO
-V^
o-f
HjN*
^ /
C.sH.gNzOsS
MW366.39
\M+HY = 367.39
i "''t
H,C
H,C
H,N*
\ /
C,5H,gN204S
MW322.38
[M+H]" = 323.38
HO
H3C
H3C
o
CsHgNO^S
in/zl90.19
CisH.eNzOsS
MW348.37
[M+H]" = 349.38
+
HN*
^ /
C.oHtNOj
MW173.17
[M+Hr= 174.18
HO,
HoC
NH*
^t<r°
C8H9O5S
MW231.22
[M+H]* = 232.23
Figure B-24: The interpretation of the positive HPLC/ESI-MS/MS of compound 366b-
isomer of penicillin G sulfone.
167
168
366c - RT26.19 min / HPLC - ( + ) - ESI - MS / MS
CsH.gNiOsS
MW366.39
[M+H]* = 367.39
r\
C6H,N02S
MW159.20
[M+H]* = 160.21
C.zHsNOjS
MW231.27
\M+HY = 232.28
. CsHieNzOsS
MW348.37
[M+H]" = 349.38
H3C^V5=N*
CjHgNS
m/zl 14.18
N CH,
CHj
r\
hX
C10H9NO4
MW207.18
[M+Hr = 208.19
C,oH,N02
MW175.19
[M+H]*= 176.19
'^Hj
/ ^
CsHuNjOjS
MW334.34
[M+H]* = 335.35
Figure B-26: The interpretation of the positive HPLC/ESI-MS/MS of compound 366c-
isomer of peniciUin G sulfone.
169
souepunqv 9A|)e|9^
a3uepunqv 9A||e|3y
o
G
•a
a,
o
o
M
I
(J
o
I
o
u
o
00
czi
00
u
h-1
>
el
(U
H
CQ
00
170
366c - RT26.25 min / HPLC - ( - ) ESI - MS / MS
/
°. X
HO
CisHigNzOsS
MW366.39
[M-H]- = 365.38
/ %
CHa
H3
CsHmNzOjS
MW292.39
[M-H]- = 291.39
CjHitNOzS
MW251.34
[M-H]- = 250.33
H,c
C6H9NO2S
MWl 59.20
[M-H]-= 158.19
/ ^
CH,
CH,
0-
CnHieNjOjS
MW288.36
[M-H]- = 287.36
CH,
//\
CH,
Cl6H,6N204S
MW332.37
[M-H]- = 331.36
Figure B-28: The interpretation of the negative HPLC/ESI-MS/MS of compound 366c-
isomer of penicilhn G sulfone.
.1 ■■
t ■
171
souepunqv sAjieia^
. . 2ooo
u. Et-oo
Zco I— ULCO^tT
t = cnoo
■ 3 o T- oj
o> o o o d> o
O CO ID •* CM
aouepunqv 9A!)E|9^
1/
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i-
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o
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00
00
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00
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H-1
CO
O
o
CQ
o
so
172
368 - RTl 1.64 min - HPLC / ( + ) ESI - MS / MS
CeHsNOjS
MWl 59.20
[M+Hr= 160.21
//
o
C,4H,3N203S
ni/z289.33
.s:!=-=CH,
CisHjoNiOeS
MW368.40
[M+Hr = 369.41
C,5H2oN204S
MW324.39
[M+H]^ = 325.40
CsH.gNjOjS
MW306.38
[M+Hr= 307.39
Figure B-30: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicilloic
acid sulfoxide.
''-yil-'i}r'..
APPENDIX C
THE PRODUCTS OF RECOMBINATION REACTIONS OF PENICILLIN G
FOLLOWING THE INTERACTION WITH POTASSIUM SUPEROXIDE - THE
CHROMATOGRAMS AND MASS SPECTRA OF HPLC/ESI-MS/MS.
o
■0-
CM
o
<s>
a.
SS8
ffiSp
*? CO 3O
loKo
Oc^O
oinO
J <mo
S>"°
-»So
<D<isa
1.41E6
= 100.0-3
-ull ms2
00 - 420
.32E
100
ull m
0-4
60E
100.
jll m
0-4
-- u U-H
- Iiu-C!
i^fi
^f?2.
^^."S
^
.-"
5<
*?^ p>
aouepunqv eAjieia^
o in
JXITTTTT
8 S
Si
l-H
CM
(U
c
CM
g
rt
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CM 0)
C
H
<u
ex
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C/1
.<£>
t/3
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C/3
-CM
tiJ
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s
c>
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OT
-<o
cx
lU
U
aouepunqv aAtiep^
b
174
175
296 - RT4.39 min - HPLC / ( + ) ESI - MS / MS
H3C-
Hjn:
CH3 s-
/
-s
OH
-CH,
NH,
CH,
OH
C10H20N2O4S2
MW296.40
[M+Hr = 297.41
H3C-
CH3 s
NH,
OH
CsHioNOiSj
m/zl 80.26
H3C
=s=s
C4S5NOS2
MW147.21
[M+H]^ = 148.22
C,H,3N202S2
ni/2245.33
H3C
H3C
1
C3H7S2
m/zl 07.21
H3C-
H2N:
CH3 s-
/
-S
OH
-CH,
CH,
OH
C,oH,7N04S2
MW279.37
[M+H]* = 280.37
H3C-
O^
H2N;
CH3 s-
/
-s
OH
-CH,
CH,
CoHisNOjSz
MW261.35
[M+H]^ = 262.36
Figure C-2: The interpretation of the positive HPLC/ESI-MS/MS of penicillamine dimer.
176
o
o
!■
o
o
o
GO
I
CO
O
_>
O
cx
<L>
H
I
U
P
b
asuepunqv SAi|e|3y
177
440 - RT24.55 min - HPLC / ( + ) ESI - MS /MS
C20H20N2O3
MW336.39
[M+Uy = 337.34
OHO
C22H20N2O6S
MW440.47
[M+H]+ = 441.48
C2,H,6N205S
MW408.43
[M+H]*= 409.43
o^>
0:
OH S^
OH
C7H8N2O6S
MW248.22
[M+H]^ = 249.22
HX
H
o h=^
//
OH
CgHioNCS
in/z232.23
■s^=CHj
H3C
H3C >=0
■^ Si=CH2
CgHioNOjS
m/z200.23
Figure C-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 440.
178
rf
93uepunqv 3Ai)e|3^
,.. Booo
LL tOOO
jio O^
J5
rs
1 1 ' " I " " I ' 1 1 1 1 . 1 . . I
3 Q O O C
* <D lO ^ (»
eauepunqv SAiieia^
m E
o
t
o
o
o
(/3
CO
I
h- (
00
U
a
(U
>
U3
o
I
U
b
179
632 - RT29.35 min - HPLC / ( + ) ESI - MS / MS
MH HN — ( H3C-
\ (^ \ — CH^Ha NH3
0=/ SH3C
OH
C26H40N4O8S3
MW632.80
[M+H]*= 633.81
CH,
HjN
-CH,
S\.
H3C-
Q6H38N4O7S3
MW6 14.79
[M+H]* = 615.79
CH, NH3*
C10H20N2O4S2
MW296.40
[M+H]" = 297.41
HJH*-( H,c-
HjC— ^ y-CH^Hj NHj
SHjC
CnH33N305S3
MW455.64
[M+H]* = 456.65
H^Hj nh;
SHjC
C,7H27N304S3
MW433.60
[M+H]" = 434.60
Figure C-6: The interpretation of the positive HPLC/ESI-MS/MS of compound 632.
180
s
A
«
SO
■* in
cn S
^^1
-"1
Zri 6
asuepunqv 3A||I:|3u
o
I
o
o
o
00
00
00
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H-1
>
00
ID
H
6
bO
181
632 - RT29.20 min - HPLC / ( - ) ESI - MS / MS
>-^ >-"
O ) ( )— CHpH, NH,
0=( SHjC
O'
C26H4oN408S3
MW632.80
[M-H]- = 631.79
V— NH HN ( H»C-H ('
o V— ( )~-<:hph,
0=<^ SM,C
O
C26H3,N407S3
MW614.79
[M-H]- = 613.78
m HN — ( H,c— ^ '
O \— ( )— CHpH,
C25H40N4O7S3
MW588.79
[M-H]- = 587.78
O \ ^ )— CHpH,
0=^ SHjC
C25H3,N405S3
MW570.78
[M-H]- = 569.77
C,6H2oN204S
MW336.40
[M-H]- = 335.30
C20H23N3O5S2
MW451.53
[M-H]- = 450.53
9"=
HN „
NH HN ( CHj
0=< SHjC
C22H3,N305S3
MW517.71
[M-H]-= 516.71
+
CH, NH,
C10H20N2O4S2
MW296.40
[M-H]- = 295.39
^■.
t
CH.
C6H,5NOS2
MW181.31
[M-H]- =180.31
H,C CH,
C2,H3,N304S3
MW485.67
[M-H]- = 484.66
Figure C-8: The interpretation of the negative HPLC/ESI-MS/MS of compound 632.
■ '4'
182
CM
O
o
Q.
Bo
roZ CM £
s/
(0
g»
CD
— I 9°
g?o^ ■
gf
o
T"i 1 1 1 F I [ n 1 1 [ 1 1
o o o
CD lO TT
i|i"i|iiiiim
o
-m
oouepunqv aAfiejay
UJ u_ Eooo
Zoj h-Li.(£iT-r^
^'
■■^■■■■^■■■■.^'
aouepunqv 9Aj)e|9^
t I I I T r ITT p TT T T'TT t f J T I 1 I
CO
CO
>
o
ex,
H
I
U
I
183
642 - RT30.13 min - HPLC / ( + ) ESI - MS / MS
C3,H38N407S2
MW642.78
[M+H]^ = 643.79
C.sHjoNjOjS
MW308.39
[M+H]^ = 309.40
C22H33N3OSS2
MW483.64
[M+Hr= 484.65
C22H3,N304S2
MW465.62
[M+H]* = 466.63
Figure C-10: The interpretation of the positive HPLC/ESI-MS/MS of compound 642
■■' >
OW^A
184
aDuepunqv aAiie|9^
O
a
o
o
o
on
00
en
pq
O
H-1
>
U
185
642 - RT30.44 min - HPLC / ( - ) ESI - MS / MS
C3,H38N407S2
MW642.78
[M-H]- = 641.78
s — s
V
C30H38N4O5S2
MW598.77
[M-H]- = 597.76
C31H36N4O6S2
MW624.77
[M-H]- = 623.76
H,N
%
r^
C,8H,9N304S2
MW405.48
[M-H]- = 404.48
Figure C-12: The interpretation of the negative HPLC/ESI-MS/MS of compound 642.
APPENDIX D
THE PRODUCTS OF CHEMILUMINESCENT REACTION OF
PENICILLIN G FOLLOWING THE INTERACTION WITH POTASSIUM
SUPEROXIDE - THE CHROMATOGRAMS AND MASS SPECTRA OF
HPLC/ESI (OR APCI)-MS/MS.
+
o
a.
o
p
^§
■<D
OTT
mo:
Q.0O H"
r?
OS
E
t =
ID =)
C£) O
^_
-o
(D
O)-
lO
O
m
-«)
s-
lO
rj:
in-
o
<D
00
■<t
lO
(D-
•o-
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3000000000<
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187
188
122 - RT1.71 min - HPLC / ( - ) APCI - MS
// \J/
o
O"
C7H502
MW122.12
[M-H]-= 121.11
Figure D-2: The interpretation of the negative HPLC/APCI-MS of benzoic acid.
.r- a
189
C-\LCQ\dala\Sp«c\SEQ 1 979m
nw chase (NH40AC -> MeOH) C18 HPLCA*)ESI-MS;MS
S# MMa-i:ib ; R T : 33..tt>^.fe 7 AV: 10 NL 1.5<E6
F- ♦ c Fu« ms2 337.00 ( 90.00 - 420.00)
10/01/97 13:32:33
P<3 ♦ K02. 1 mfl/mL
'zio' ' '2^0' ' '260' ' '2to' ' '3A0' ' '3io' ' 'sio' ' '3^0' ' 'sio'
m/z
Rr:J2.G9. 36.12
10CH
7*:
10^
Nl;
2.92E«
TlCFz + c
FUiimZ
337.00 (
90.00-
420.001
33^2 _ 34.24 3^.37
328 330 33.2 3^.4 3^.6 ' 'lix ' 'M ' ii^' ' 'zi.* ' 3^.6'
TliT»{mIn)
_J476^^^ 35.11 35.,24 35.41 3i.56 35^1 M»1
'3^.e' ' 3io' ' '3i2' ' '3i4' ' '3i.s ' 'six ' 'sio'
C:\LCQ\dala\Spec\SEQ1979o
rev phase (NH40Ac -> MeOH) C18 HPtC/(-)eSI-MS/MS
10/01/97 15:47:44
PG ♦ K02, 1 mfl/mL
SH: 1137-1343 RT; 33 52-33 67 AV: 7 NL: 9J7E4
F: - c Fun ms2 335 10 ( 90.00 - 42O.0O1
80
8 70-=
c
«
■o 60-
I 50
0)
i 40
J2
S. 30
20-
Mf^
158
^
31S.
335
2io' ' '2io' ' 2^' ' '24o' ' '3io' ' 3^0' ' Mo' ' 'i^' ' 'sio' ' '460' ' '4io
m/z
100 120 140 160 1
RT; 32.21 ■ 35,29
lOH
•0-
70-
32.<3 3256 .32,'3 32.M 33.10 3120 33.
32 4 32 6 32 e 33
f.
NU
2.60E5
TlCF:-e
FuiimZ
U9.10I
M.00-
420.001
2' ■ d.4' ■ '3i.6' ■ '3^.8 3^.0' U.i ' '3i.*~ Ut ' ' iit' '"'j^o' ' 'jij'
TlnxtnWt)
Figure D-3: The positive and negative HPLC/ESI-MS/MS of compound 336.
.-iSf.;
190
336 - RT33.59 min - HPLC / ( + ) ESI - MS / MS
H3C.
CsHnNaOsS
MW336.36
[M+Hr= 337.37
w
s-^
nh;
x;Hj
C7H8N2O3S
MW200.21
[M+Hf = 201.22
^\
H,C-
\)
HjN
CHuNOjS
MWl 89.23
[M+Hr= 190.24
H,C
C,5H,4N204S
MW3 18.34
[M+Hr = 319.35
HX
CuHmNzOj
MW2 18.25
[M+Hr = 219.26
C8H,N0
MW135.16
[M+Hr= 136.17
Figure D-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 336.
191
336 - RT33.57 min - HPLC / ( - ) ESI - MS / MS
H,C
H,C
.^-^
CftHsNOzS
MW159.20
[M-H]-= 158.19
H3C
-=0
-^
C,5H,4N204S
MW3 18.34
[M-H]-= 317.34
CuHuNjOj
MW218.25
[M-H]= 217.25
NH .«-
CsH.iNOjS
MW177.22
[M-H]-= 176.21
\
H,C
V
C.sH.sNzOs:
MW336.36
[M-H]- = 335.35
s
//
C7H8N2O3S
MW200.21
[M-H]- = 199.20
158.19-.^^ 176.21 *-
-H2O
317.34
199.20 - '"^^' '^"^ (231.24)^
Figure D-5: The interpretation of the negative HPLC/ESI-MS/MS of compound 336.
'i yS>'' s t ' ■
192
aouepunqv 9Ai)e|3^
T- (r 1 1 1 1 1 1 1 1 II 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rin
oiQOOOQQOOO
93uepunqv sAiietdy
193
51 1 - RT31.07 min - HPLC / ( + ) ESI - MS / MS
C2SH25N3O7S
MW5 11.55
[M+H]* = 512.55
>* 'i:
CoHtNOi
MW173.17
[M+H]*= 174.18
C25H23N3O6S
MW493.53
[M+H]* = 494.54
HaC^
CgHieNaOa
MW308.33
[M+H]* = 309.34
Figure D-7: The interpretation of the positive HPLC/ESI-MS/MS of compound 511.
APPENDIX E
THE PRODUCTS OF UNKNOWN REACTIONS OF PENICILLIN G
FOLLOWING THE INTERACTION WITH POTASSIUM SUPEROXIDE - THE
CHROMATOGRAMS AND MASS SPECTRA OF HPLC/ESI-MS/MS.
. ;. i
o
CO
o
E
O)
E
O
o
Q.
•i^
<6
O
05
w o
ii)0
roZ
Is
0(0
|iii'|iiii|ii"l""|iiii|ii M |iiii|iii i | n iii nn
"ooooo--"^--
Oi CO t~ <o tn
1 1 1 1 1 1
o o o
CO CM 1-
(OU.
aouepunqv 8/M)B|8a
UJ U.<Mo
^
;;
a^
ON
O
^ fi
o
o
00
I
I— I
00
u
>
60
u
(=1
u
aouepunqv SAi)e|3y
At,
196
269 - RT22.69 min. - HPLC / ( - ) ESI - MS / MS
S— CH,
// ^
C,2H,5N04S
MW269.31
[M-H]- = 268.31
/ \
u
-
^\
CnH
UNO3S
MW237.27
[M-H]-
= 236.26
O N
A
S — CH,
//
C5H7NO4
MW177.17
[M-H]- =176.17
N
f
=S=CH"
CSH3NO2S
MW141.14
[M-H]-= 140.14
Figure E-2: The interpretation of the negative HPLC/ESI-MS/MS of compound 269.
•V ..;
i >
197
o
o
CO-
^
o
-CM
CO
CO
O
O
CO
o
I?
a>
So
CO o
oO
a.-t
Wi
(0
■o
o 2
r
o
. . + '"
It ..
(Ou.
1 1] in I |i 1 1 1 1 1 1 1 1 |ni i| M III I III 1 1 1
----- o o
souepunqv sAiteis^
o
-co N
'1
U-Oo
OPCM
UJ u."o
ZcDH Era
"-go
S,i:"88'
2(D I- EcoTf
«i
o E
F
s
[,, o CO S -^ csj
souepunqv SAqep^
O
&,
B
o
o
o
C/3
00
I
I— (
w
u
h-1
(U
>
• i-H
o
a,
u
H
tn
I
W
§
GO
198
353 - RT39.43 / HPLC - ( + ) ESI - MS /MS
OH
A
-CH,
CH,
C,6H„N06S
MW353.39
[M+H]* = 354.39
OH O
CsHnNOsS
MW335.37
[M+H]* = 336.38
OHjO^
OH
CH,
C7H10O5S
MW206.21
[M+H]* = 207.22
\
-CH,
CH,
C7H10O3S
MWl 74.21
[M+Hr= 175.22
' OH
H2C,
r
-CH,
CH,
CeHiiOsS
m/zl63.21
CH,
CH,
C4H7OS
m/zl03.16
Figure E-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 353.
h J V
,'.ff .■-"W^f"" ■■ 'VJ* '
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